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

What is Ccdc90b and what is its cellular localization?

Ccdc90b (Coiled-coil domain-containing protein 90B) is a mitochondrial protein encoded by the Ccdc90b gene in mice. The protein is characterized by a distinctive head-neck-stalk-anchor architecture common to a heterogeneous group of trimeric membrane-anchored proteins found in both prokaryotes and eukaryotic organelles . Cellular localization studies confirm that Ccdc90b is primarily targeted to mitochondria, as it contains a specific mitochondrial targeting sequence at its N-terminus . The protein is part of a class of membrane-bound coiled-coil proteins that function as important mediators of signaling, fusion, and scaffolding within the mitochondrial environment .

What is the molecular structure of mouse Ccdc90b?

Mouse Ccdc90b exhibits a characteristic head-neck-stalk-anchor architecture where:

• The N-terminal head domain is connected to a coiled-coil stalk via a β-layer neck region

• The stalk is anchored to the membrane

• The protein contains seven predicted alpha helices, which is typical of coiled-coil proteins

• The protein contains a domain of unknown function (DUF1640) that characterizes most of the protein with the exception of the first 23 amino acid residues

Crystal structure studies of the human CCDC90B (a homolog of mouse Ccdc90b) have revealed that the conserved head domain serves as a mediator of its function, particularly in interaction with the mitochondrial calcium uniporter (MCU) .

What are the key physiochemical properties of Ccdc90b?

Ccdc90b has the following physiochemical properties:

The protein contains a mitochondrial targeting sequence (MNSRQAWRLFLSQGRGDRWVSRP) at the first 23 amino acid residues, which is cleaved after targeting to mitochondria . This targeting sequence is essential for proper localization and function of the protein within the cellular environment.

What post-translational modifications have been identified in Ccdc90b?

Several post-translational modifications have been identified in mouse Ccdc90b:

• Acetylation: Documented at lysine residues K167 and K197 according to PhosphoSitePlus database

• Phosphorylation sites: Ccdc90b is predicted to contain at least three specific phosphorylation sites:

  • Protein Kinase C phosphorylation sites

  • Casein Kinase II phosphorylation sites

  • cAMP/cGMP dependent phosphorylation sites

• Mitochondrial targeting sequence processing: The N-terminal 23 amino acid residues (MNSRQAWRLFLSQGRGDRWVSRP) function as a mitochondrial targeting signal that is cleaved after import into mitochondria

The protein does not appear to undergo other major modifications such as chloroplast transit peptide processing, signal peptide cleavage, C-mannosylation, or N-glycosylation based on prediction algorithms .

How is the mitochondrial targeting sequence of Ccdc90b processed?

The mitochondrial targeting of Ccdc90b occurs through a defined sequence of events:

• The first 23 amino acid residues (MNSRQAWRLFLSQGRGDRWVSRP) at the N-terminus serve as the mitochondrial targeting signal

• This presequence is recognized by receptors on the outer mitochondrial membrane, which facilitate import through the translocase of the outer membrane (TOM) complex

• Further transport through the inner membrane occurs via the translocase of the inner membrane (TIM) complex

• Once inside the mitochondrial matrix, the targeting sequence is cleaved by the mitochondrial processing peptidase (MPP)

• The mature protein then assumes its functional conformation and localization within the mitochondria

This processing is essential for proper function, as the cleaved mature protein is the active form that participates in mitochondrial processes.

What are the known protein interaction partners of Ccdc90b?

Ccdc90b interacts with several proteins as detected by various experimental methods including yeast two-hybrid, co-immunoprecipitation, and pull-down assays. Key interaction partners include:

• Mitochondrial Calcium Uniporter (MCU): Ccdc90b's head domain directly interacts with MCU, suggesting a role in calcium handling

• Other documented interaction partners:

  • TK1 (Thymidine Kinase 1)

  • TSC22D1 (TSC22 Domain Family Member 1)

  • PSMD11 (26S Proteasome Non-ATPase Regulatory Subunit 11)

  • KDM1A (Lysine Demethylase 1A)

  • PRDX5 (Peroxiredoxin 5)

  • CDKN2B (Cyclin Dependent Kinase Inhibitor 2B)

  • CDKN2C (Cyclin Dependent Kinase Inhibitor 2C)

  • PIN1 (Peptidyl-Prolyl Cis-Trans Isomerase NIMA-Interacting 1)

  • NACA (Nascent Polypeptide-Associated Complex Alpha Subunit)

These interactions suggest that Ccdc90b may be involved in diverse cellular processes beyond its primary mitochondrial function, potentially including cell cycle regulation, protein quality control, and redox homeostasis.

How does Ccdc90b functionally relate to MCUR1?

MCUR1 (Mitochondrial Calcium Uniporter Regulator 1) is a functionally characterized paralog of Ccdc90b. Their relationship includes:

• Structural similarity: Both proteins share the characteristic head-neck-stalk-anchor architecture

• Calcium handling: Studies using MCUR1 have shown that the head domain interacts directly with the mitochondrial calcium uniporter (MCU) and becomes destabilized upon Ca²⁺ binding

• Functional domains: Research on MCUR1 has helped elucidate the role of individual domains, which by homology may apply to Ccdc90b as well

• Evolutionary conservation: Both proteins are part of an evolutionarily conserved family of membrane-bound coiled-coil proteins with the conserved head domain serving as a mediator of function

Domain-specific studies have demonstrated that the head domain of these proteins is particularly important for the interaction with MCU, suggesting that Ccdc90b may play a similar role in mitochondrial calcium handling through its interaction with the calcium uniporter complex.

What techniques are most effective for studying Ccdc90b localization and expression?

Several techniques have proven effective for studying Ccdc90b localization and expression:

• Immunofluorescence microscopy:

  • Co-staining with mitochondrial markers (MitoTracker, TOM20, etc.)

  • Visualization of tagged Ccdc90b (GFP, FLAG, etc.) in fixed or live cells

• Subcellular fractionation:

  • Isolation of mitochondria followed by Western blotting

  • Proteinase K protection assays to determine submitochondrial localization

• Expression analysis:

  • qRT-PCR for mRNA quantification

  • Western blotting for protein level detection

  • Mass spectrometry for comprehensive proteomic analysis

• CRISPR-based approaches:

  • CRISPR activation systems for upregulation of endogenous gene expression

  • CRISPR/Cas9 for genome editing and functional studies

• Electron microscopy:

  • Immunogold labeling for precise localization within mitochondrial compartments

These techniques provide complementary information about Ccdc90b's expression patterns, subcellular localization, and potential functional roles in different tissues and experimental conditions.

How can CRISPR/Cas9 technology be utilized to study Ccdc90b function?

CRISPR/Cas9 technology offers powerful approaches for studying Ccdc90b function:

• Gene knockout (KO):

  • Complete elimination of Ccdc90b expression using CRISPR/Cas9-mediated gene disruption

  • Analysis of phenotypic consequences to understand protein function

• Gene activation:

  • CRISPR Activation (CRISPRa) systems utilizing deactivated Cas9 (dCas9) fused to VP64 activation domain

  • Synergistic activation mediator (SAM) transcription activation system to upregulate endogenous Ccdc90b expression

  • Lentiviral delivery of CRISPR activation components for stable expression

• Domain-specific mutations:

  • Targeted modification of specific domains (head, neck, stalk) to understand their functional importance

  • Creation of phosphorylation-deficient mutants to study the role of post-translational modifications

• Reporter gene knock-in:

  • Integration of fluorescent protein tags for live-cell tracking of expression and localization

  • Addition of affinity tags for protein purification and interaction studies

Commercial CRISPR systems such as CCDC90B Lentiviral Activation Particles are available for mouse (m) Ccdc90b gene, which employ the SAM transcription activation system designed to specifically upregulate expression of the Ccdc90b gene via lentiviral transduction .

What is the role of Ccdc90b in mitochondrial calcium homeostasis?

Ccdc90b's role in mitochondrial calcium homeostasis appears to be closely linked to the mitochondrial calcium uniporter (MCU) complex:

• Direct interaction with MCU: Crystallographic and functional studies indicate that Ccdc90b's head domain interacts directly with MCU

• Calcium sensitivity: The head domain is destabilized upon Ca²⁺ binding, suggesting a potential regulatory mechanism in response to calcium concentrations

• Structural implications: The trimeric membrane-anchored architecture of Ccdc90b positions it to potentially influence calcium channel activity or assembly

• Regulatory function: By analogy to its paralog MCUR1, Ccdc90b may serve as a regulator of MCU activity, potentially influencing calcium uptake or channel properties

The calcium-dependent destabilization of the head domain suggests a potential feedback mechanism, where changes in calcium levels could alter Ccdc90b's interaction with MCU, thereby modulating calcium uptake into mitochondria. This represents a sophisticated regulatory circuit for maintaining mitochondrial calcium homeostasis, which is critical for numerous cellular processes including energy production, apoptosis, and signaling.

What are the current challenges in studying the function of Ccdc90b?

Researchers face several challenges when investigating Ccdc90b:

• Functional redundancy: Potential overlapping functions with its paralog MCUR1 may mask phenotypes in single knockout models

• Tissue-specific expression: Varying expression patterns across tissues may necessitate tissue-specific studies

• Technical challenges:

  • Maintaining proper protein folding and mitochondrial targeting in recombinant expression systems

  • Difficulty in crystallizing membrane-associated proteins for structural studies

  • Limited availability of specific antibodies for mouse Ccdc90b

• Complex interactions:

  • Interactions with multiple proteins (as evidenced by documented protein-protein interactions)

  • Potential involvement in diverse cellular pathways beyond calcium handling

• Transient or dynamic interactions:

  • Calcium-dependent interactions may be difficult to capture in standard experimental conditions

  • Post-translational modifications may alter interaction patterns in context-dependent ways

Addressing these challenges requires integrated approaches combining genetics, biochemistry, structural biology, and advanced imaging techniques to fully elucidate Ccdc90b's role in mitochondrial function and broader cellular processes.

How conserved is Ccdc90b across species and what does this suggest about its function?

Ccdc90b shows notable evolutionary conservation across species, providing insights into its fundamental function:

• Cross-species availability: Recombinant Ccdc90b proteins are available from multiple species including:

  • Human

  • Mouse

  • Rat

  • Rhesus macaque

  • Zebrafish

  • Xenopus tropicalis (Western clawed frog)

• Structural conservation: The characteristic head-neck-stalk-anchor architecture is observed in both prokaryotes and eukaryotic organelles, suggesting an ancient and fundamental role

• Domain conservation: The DUF1640 domain that characterizes the protein is maintained across species, indicating functional importance

• Head domain importance: The conserved head domain has been identified as a mediator of function across this protein class

The high degree of conservation suggests that Ccdc90b likely serves a fundamental role in mitochondrial biology that has been maintained throughout evolution. The presence of similar structural features in both prokaryotic and eukaryotic proteins points to a possible endosymbiotic origin, consistent with the evolutionary history of mitochondria.

What are the key differences between mouse and human CCDC90B?

While mouse Ccdc90b and human CCDC90B share significant similarities, several notable differences exist:

• Chromosomal location:

  • Human: Chromosome 11

  • Mouse: Chromosome 7 E1

• Neighboring genes:

  • Human CCDC90B is neighbored by PCF11, ANKRD42, BC070093, and DLG2

  • Mouse Ccdc90b may have different genomic context

• Interacting partners:

  • While core interactions (such as with MCU) are likely conserved, species-specific interaction partners may exist

  • Regulatory mechanisms may differ between species

• Expression patterns:

  • Tissue-specific expression levels may vary between mouse and human

  • Developmental regulation may show species-specific patterns

Despite these differences, the fundamental structural features and mitochondrial localization are conserved, suggesting that mouse models remain valuable for understanding the basic function of CCDC90B in human physiology and disease.