Recombinant Mouse Coiled-coil domain-containing protein 93 (Ccdc93)

What is the basic structure of CCDC93 protein and how does it compare between humans and mice?

CCDC93 is a structural protein characterized by coiled-coil domains. Human CCDC93 features a bimodular structure with an N-terminal NDC80/NUF2-calponin homology (NN-CH) domain and a C-terminal coiled-coil domain. Mouse CCDC93 shares significant sequence homology with human CCDC93, particularly in the C-terminal region with approximately 55% amino acid similarity . Notably, unlike human CCDC93, mouse CCDC93 lacks the NN-CH domain, which may suggest functional adaptations specific to each species . The protein's structural features are critical for its interactions with other components of the CCC (COMMD/CCDC22/CCDC93) complex, including its binding to COMMD proteins primarily through the COMM domain .

What is the main function of CCDC93 in cellular processes?

CCDC93 functions primarily in endosomal trafficking as a key component of the CCC (COMMD/CCDC22/CCDC93) complex. This complex works in close association with the retriever complex to facilitate the recycling of endocytosed integral plasma membrane proteins from early endosomes back to the plasma membrane . Specifically, CCDC93 participates in maintaining proper endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P), which is critical for endosomal sorting . In mice, CCDC93 has been demonstrated to influence LDL receptor (LDLR) recycling, with implications for plasma LDL cholesterol levels . Additionally, CCDC93 plays essential roles in embryonic development, as homozygous deletion in mice results in embryonic lethality before day E10.5 .

How does CCDC93 interact with other proteins in the endosomal trafficking system?

CCDC93 forms a complex with CCDC22 and COMMD proteins to create the CCC complex, which functions alongside but distinct from the retriever complex . Biochemical studies have shown that:

• CCDC93 directly interacts with multiple COMMD family proteins (including COMMD1, 7, 9, and 10) primarily through the COMM domain

• The binding affinity between CCDC93's NN-CH domain and the COMM domain of COMMD1 has been quantified at approximately 14.6 μM

• While closely associated with the retriever complex (consisting of VPS35L, VPS26C, and VPS29), the CCC complex appears to be a distinct molecular assembly as demonstrated by blue native gel separation techniques

• CCDC93 stability is interdependent with other CCC components, as deletion of COMMD3 leads to significant reduction in CCDC93 and CCDC22 protein levels

These interactions facilitate the selective recycling of specific membrane proteins and regulate the endosomal environment.

What are the most effective methods for generating Ccdc93 knockout mouse models?

Generation of viable Ccdc93 knockout models requires careful consideration of the embryonic lethality observed in homozygous knockouts. The most successful approaches include:

• CRISPR/Cas9-mediated genome editing targeting specific exons of the Ccdc93 gene, as demonstrated in studies examining the relationship between CCDC93 and blood pressure regulation . For example, one effective approach utilized CRISPR/Cas9 to create a 21-nucleotide deletion spanning intron 6/exon 7 (2 nucleotides in intron 6 and 19 nucleotides in exon 7) .

• Heterozygous (Ccdc93+/-) mouse models are viable and morphologically normal while showing approximately 1.3-fold lower aortic Ccdc93 protein expression (P = 0.0041) compared to wild-type littermates, making them suitable for functional studies .

• For embryonic studies, timed mating between Ccdc93+/- mice allows for examination of embryos at specific developmental stages (e.g., E10.5) to assess the impact of complete Ccdc93 deficiency on embryonic development .

• Tissue-specific conditional knockout models may be preferable for studying CCDC93 function in specific cell types while avoiding embryonic lethality, though these advanced models were not explicitly described in the search results.

Validation of knockout efficiency should include both mRNA and protein quantification using qRT-PCR and western blotting with appropriate antibodies against Ccdc93.

What are the recommended approaches for evaluating CCDC93's role in endosomal trafficking?

To effectively investigate CCDC93's function in endosomal trafficking, researchers should consider these methodological approaches:

• Protein localization studies using fluorescently tagged CCDC93 (e.g., CCDC93-RFP fusion proteins) to visualize its distribution within endosomal compartments .

• Co-immunoprecipitation followed by mass spectrometry to identify CCDC93-interacting proteins, as demonstrated in studies that revealed interactions between CCDC93 and components of both the CCC and retriever complexes .

• Blue native gel electrophoresis to determine the size and composition of native protein complexes containing CCDC93, which has been useful in distinguishing between the CCC and retriever complexes .

• Functional trafficking assays measuring recycling of known cargoes such as β1 integrin, LDLR, and LRP1, which are dependent on CCC complex function .

• Quantification of endosomal PI(3)P levels using specific probes, as CCDC93 has been implicated in maintaining normal endosomal PI(3)P levels .

• Genetic interaction studies examining double mutants (e.g., vti13 ccdc93 in Arabidopsis) to identify functional relationships between CCDC93 and other components of trafficking pathways .

What techniques are most reliable for measuring CCDC93 protein stability and expression levels?

Based on published research methodologies, the following techniques provide reliable measurements of CCDC93 protein stability and expression:

• Quantitative western blotting using CCDC93-specific antibodies with normalization to appropriate housekeeping proteins. This approach has successfully demonstrated reduced Ccdc93 protein expression in heterozygous mouse models (approximately 1.3-fold reduction compared to wild-type) .

• qRT-PCR for mRNA quantification using validated primers specific to Ccdc93. For example, researchers have used primers targeting specific regions of Ccdc93 transcript to confirm knockdown efficiency in mutant models .

• Pulse-chase experiments to measure protein half-life and stability. Studies have shown that specific variants (e.g., Pro228Leu) can increase CCDC93 protein stability , suggesting this approach is valuable for characterizing the effects of mutations.

• Expression QTL (eQTL) analysis to correlate genetic variants with expression levels. For instance, the rs33975708 variant demonstrated a significant association with lower CCDC93 expression in tibial artery tissue (P = 1.4x10-5, normalized effect size = -0.2) .

• Immunohistochemistry for tissue-specific expression patterns, which has revealed enrichment of Ccdc93 protein in the endothelium of mesenteric arteries in wild-type mice .

• Endothelial cell-specific translating ribosome affinity purification (EC-TRAP) to confirm cell type-specific expression, which demonstrated enrichment of Ccdc93 transcript in endothelial cells of thoracic aortae and mesenteric arteries .

How do genetic variants in CCDC93 affect cardiovascular health and lipid metabolism?

Research has identified several CCDC93 genetic variants with significant cardiovascular and metabolic implications:

• The CCDC93 p.Pro228Leu variant (in strong linkage disequilibrium with rs10490626, r² > 0.96) is dose-dependently associated with:

  • Lower LDL cholesterol levels (P-trend 2.5 × 10⁻⁶ to 8.0 × 10⁻⁹)

  • Lower risk of myocardial infarction (P-trend 0.04-0.002)

  • Lower risk of cardiovascular mortality (P-trend 0.005-0.004)

• The p.Pro228Leu variant increases CCDC93 protein stability, and overexpression of human CCDC93 in mice decreases plasma LDL cholesterol .

• The coding variant c.535C>T, p.Arg179Cys (rs33975708, MAF = 0.15%) is associated with increased central systolic blood pressure (β = 29.3 mmHg, P = 1.23x10⁻⁷) .

• Mechanistically, CCDC93 appears to influence cardiovascular health through:

  • Regulation of LDLR recycling, affecting plasma LDL levels

  • Maintenance of vascular function, as heterozygous Ccdc93 mice show impaired arterial relaxation and enhanced contractile responses

  • Effects on fatty acid metabolism and mitochondrial function, with Ccdc93+/- mice exhibiting elevated plasma free fatty acid levels (96±7mM vs 124±13mM, P = 0.0031) and aortic mitochondrial dysfunction

These findings position CCDC93 as an important regulator of cardiovascular health through multiple pathways including lipid metabolism, vascular function, and mitochondrial homeostasis.

What is the evidence for CCDC93's role in blood pressure regulation?

Multiple lines of evidence support CCDC93's involvement in blood pressure regulation:

• Genetic evidence: An exome-wide association study in a Han Chinese population identified the rare coding variant rs33975708 (CCDC93 c.535C>T, p.Arg179Cys, MAF = 0.15%) as strongly associated with increased central systolic blood pressure (β = 29.3 mmHg, P = 1.23x10⁻⁷) .

• Expression correlation: rs33975708 showed an expression QTL association with lower CCDC93 expression in tibial artery tissue (P = 0.000014), suggesting a potential mechanism through reduced expression .

• Animal model validation: Heterozygous Ccdc93+/- mice exhibited significantly elevated systolic blood pressure compared to wild-type littermates (125±10 mmHg vs 110±8 mmHg, P = 0.016) .

• Vascular function studies: Wire myography of Ccdc93+/- mouse aortae demonstrated:

  • Impaired acetylcholine-induced relaxation

  • Enhanced phenylephrine-induced contraction

• Molecular pathway analysis: RNA-Seq of Ccdc93+/- mouse thoracic aortae identified significantly enriched pathways in fatty acid metabolism and mitochondrial metabolism, suggesting these mechanisms may mediate CCDC93's effect on blood pressure regulation .

The convergence of human genetic association data with functional validation in animal models provides compelling evidence for CCDC93's role in blood pressure regulation through effects on vascular function and metabolism.

What is the significance of CCDC93's embryonic lethality in homozygous knockout mice?

The embryonic lethality observed in homozygous Ccdc93 knockout mice reveals several important aspects of CCDC93 biology:

• Essential developmental role: Ccdc93 null (Ccdc93-/-) mice die before embryonic day E10.5, indicating that CCDC93 is indispensable for early embryonic development .

• Timing and pattern of lethality: Timed mating experiments showed that approximately 9% of fetoplacental units at E10.5 were Ccdc93-/-, with histological analysis revealing:

  • Resorption sites containing hemorrhage and necrotic debris

  • Neutrophilic and lymphocytic inflammation within and around chorioallantoic membranes

  • Complete absence of embryos or embryonic tissues, suggesting extremely early developmental arrest or failure

• Dosage sensitivity: While heterozygous Ccdc93+/- mice are viable and morphologically normal, they exhibit significant phenotypes including:

  • Elevated blood pressure

  • Vascular dysfunction

  • Metabolic alterations

• Evolutionary conservation: The essential role of CCDC93 appears to be conserved across species, as studies in Arabidopsis also show that CCDC93 is required for normal growth .

The embryonic lethality of Ccdc93-/- mice highlights the fundamental importance of this protein in development, likely through its critical role in membrane trafficking pathways essential for early embryonic patterning and growth.

How does the CCC complex coordinate with retriever to regulate endosomal trafficking?

The relationship between the CCC complex (containing CCDC93) and retriever represents a sophisticated coordination system in endosomal trafficking:

• Structural relationship: While closely associated, CCC and retriever appear to be distinct molecular assemblies:

  • Proteomic analysis reveals overlapping interactions between components of both complexes

  • Blue native gel separation shows distinct molecular weights for CCC (~500 kDa) versus retriever (~400 kDa) complexes

  • Endogenous immunoprecipitation of CCC components leads to limited or undetectable co-precipitation of retriever-specific components

• Shared and distinct components:

  • Both complexes share VPS35L, suggesting it may serve as a bridging factor

  • The CCC complex includes COMMD proteins, CCDC22, and CCDC93

  • The retriever complex consists of VPS35L, VPS26C, and VPS29

• Functional synergy in cargo recognition and trafficking:

  • The CCC complex regulates endosomal PI(3)P levels, creating an appropriate environment for retriever function

  • Retriever (particularly through SNX17) recognizes specific cargo proteins for recycling

  • Together, they facilitate the recycling of critical membrane proteins including β1 integrin, LDLR, and LRP1

• Genetic interaction: Studies in Arabidopsis demonstrate that both VPS26C (retriever component) and CCDC93 can suppress the short root hair phenotype of vti13 mutants, suggesting functional overlap or coordination in trafficking pathways .

The emerging model suggests that while physically distinct, these complexes function in a coordinated manner where CCC (including CCDC93) establishes and maintains the appropriate endosomal environment through PI(3)P regulation, enabling retriever to efficiently recognize and sort cargo proteins.

What are the tissue-specific functions of CCDC93, particularly in cardiovascular tissues?

CCDC93 exhibits important tissue-specific functions, with particular significance in cardiovascular tissues:

• Endothelial expression pattern:

  • Immunohistochemistry has shown Ccdc93 protein expression is enriched in the endothelium of mesenteric arteries in wild-type mice

  • Endothelial cell-specific translating ribosome affinity purification (EC-TRAP) confirmed enrichment of Ccdc93 transcript in endothelial cells of thoracic aortae and mesenteric arteries

• Vascular functional impact:

  • Heterozygous Ccdc93+/- mice show impaired acetylcholine-induced relaxation and enhanced phenylephrine-induced contraction in wire myography experiments

  • These vascular changes correlate with elevated systolic blood pressure (125±10 mmHg vs 110±8 mmHg in wild-type, P = 0.016)

• Metabolic regulation in vascular tissue:

  • RNA-Seq analysis of Ccdc93+/- mouse thoracic aortae revealed significantly enriched pathways in fatty acid metabolism and mitochondrial function

  • Ccdc93+/- mice exhibit elevated plasma free fatty acid levels (124±13mM vs 96±7mM in wild-type, P = 0.0031)

  • Aortic mitochondrial dysfunction observed through aberrant Parkin and Nix protein expression

• LDL receptor regulation:

  • CCDC93 influences LDLR recycling, with genetic variants associated with altered plasma LDL cholesterol levels

  • Overexpression of human CCDC93 in mice decreases plasma LDL cholesterol

These tissue-specific functions position CCDC93 as a critical regulator of cardiovascular health through its effects on vascular function, lipid metabolism, and mitochondrial homeostasis in vascular tissues.

How do post-translational modifications affect CCDC93 function and stability?

While the search results don't provide direct information about post-translational modifications (PTMs) of CCDC93, we can infer several important aspects based on related findings:

• Genetic variants affecting stability:

  • The p.Pro228Leu variant increases CCDC93 protein stability , suggesting that amino acid changes in specific regions can significantly impact protein turnover

  • This implies potential PTM sites in this region that may regulate normal CCDC93 degradation

• Complex formation dependencies:

  • CCDC93 protein levels are reduced upon deletion of other CCC complex components (e.g., COMMD3)

  • This interdependence suggests that complex formation may protect CCDC93 from degradation, possibly by masking PTM sites that would otherwise target it for degradation

• Potential phosphorylation:

  • CCDC93's role in PI(3)P regulation implies possible interactions with kinases and phosphatases

  • The protein likely contains phosphorylation sites that could regulate its function or interactions within the endosomal environment

• Evolutionary differences:

  • The structural differences between human and plant CCDC93 (e.g., loss of NN-CH domain in Arabidopsis) suggest that PTM patterns may also differ across species, potentially explaining functional adaptations

Advanced research in this area would benefit from:

• Phosphoproteomic analysis of CCDC93 under various cellular conditions

• Site-directed mutagenesis of potential PTM sites

• Investigation of ubiquitination patterns that might regulate CCDC93 turnover

• Identification of enzymes that catalyze PTMs on CCDC93

What are the emerging therapeutic implications of targeting CCDC93 pathways for cardiovascular diseases?

Based on current research findings, targeting CCDC93 pathways presents several promising therapeutic avenues for cardiovascular diseases:

• LDL cholesterol regulation:

  • The association between CCDC93 variants and LDL cholesterol levels suggests that enhancing CCDC93 function could provide a novel approach to reduce LDL cholesterol

  • Given that the p.Pro228Leu variant increases CCDC93 protein stability and is associated with lower LDL-c levels , small molecules that stabilize CCDC93 protein could potentially mimic this beneficial effect

• Blood pressure regulation:

  • The link between CCDC93 variants and central systolic blood pressure suggests that modulating CCDC93 expression or function could represent a new target for antihypertensive therapy

  • Compounds that enhance CCDC93 expression or activity might counteract the effects of loss-of-function variants associated with elevated blood pressure

• Vascular function improvement:

  • The impaired arterial relaxation and enhanced contractile responses observed in Ccdc93+/- mice indicate that CCDC93 modulators could potentially improve vascular function

  • This might be particularly relevant for conditions characterized by endothelial dysfunction

• Mitochondrial metabolism:

  • The connection between CCDC93 and mitochondrial function opens possibilities for targeting metabolic aspects of cardiovascular disease

  • Compounds that address the aberrant fatty acid metabolism observed in Ccdc93+/- mice could provide metabolic support in conjunction with CCDC93 pathway modulation

• Endosomal trafficking:

  • Since CCDC93 functions in endosomal PI(3)P regulation and protein recycling , targeted approaches to enhance these cellular processes could restore normal trafficking of key cardiovascular receptors like LDLR

While direct therapeutic applications remain to be developed, the multifaceted role of CCDC93 in cardiovascular biology provides a rich landscape for drug discovery efforts focused on novel mechanisms distinct from current cardiovascular therapies.

What are the best expression systems for producing recombinant mouse CCDC93 protein?

For optimal production of recombinant mouse CCDC93 protein, researchers should consider these technical approaches based on published methodologies:

• Mammalian expression systems:

  • HEK293T cells have been successfully used for expression of CCDC93 with appropriate tags (e.g., HA-tagged CCDC93)

  • This system provides proper folding and potential post-translational modifications relevant to mammalian CCDC93

• Bacterial expression strategies:

  • For structural studies, expression of specific domains (e.g., the NN-CH domain) rather than full-length protein may improve solubility

  • GST-fusion proteins have been successfully used for pull-down experiments with CCDC93 domains

• Co-expression considerations:

  • Co-expression with interaction partners (e.g., CCDC22 or COMMD proteins) may improve stability and solubility of recombinant CCDC93

  • Research indicates that interactions between COMMD proteins and CCDC93 occur spontaneously in vitro , suggesting potential benefits of co-purification approaches

• Purification tags and conditions:

  • N-terminal tags appear preferable as C-terminal tags may interfere with coiled-coil domain functions

  • Affinity tags followed by size exclusion chromatography have yielded preparations suitable for binding assays such as biolayer interferometry

• Storage and stability:

  • While specific information for CCDC93 is not provided in the search results, proteins with coiled-coil domains typically benefit from storage buffers containing glycerol and reducing agents

  • Avoiding repeated freeze-thaw cycles is generally recommended for maintaining protein function

For functional studies, it's important to validate that recombinant CCDC93 retains its ability to interact with known binding partners through appropriate binding assays.

What challenges exist in studying CCDC93's embryonic functions given the lethality of homozygous knockout?

Investigating CCDC93's embryonic functions faces several methodological challenges due to the early lethality of homozygous knockouts:

• Temporal limitations:

  • Ccdc93-/- embryos die before E10.5, restricting analysis to very early developmental stages

  • This requires precise timed mating protocols and rapid collection of embryos before resorption

• Technical approaches to overcome lethality:

  • Conditional knockout strategies using tissue-specific or inducible Cre-lox systems would allow for targeted deletion in specific tissues or developmental stages

  • Partial knockdown approaches (e.g., using shRNA or hypomorphic alleles) might allow for examining dose-dependent effects while avoiding complete lethality

• Ex vivo and in vitro alternatives:

  • Embryonic stem cell (ESC) models derived from Ccdc93+/- mice could be used to generate Ccdc93-/- cell lines for in vitro differentiation studies

  • Organ explant cultures from early embryos prior to lethality could provide insights into tissue-specific functions

• Chimeric approaches:

  • Generating chimeric embryos with varying contributions of Ccdc93-/- cells could help identify critical tissues and cell types where CCDC93 function is essential

• Cross-species comparative studies:

  • Utilizing model organisms where CCDC93 knockdown may not be lethal (e.g., Arabidopsis studies show viable plants with CCDC93 mutations )

  • This could provide indirect insights into conserved functions relevant to mammalian development

• Rescue experiments:

  • Testing whether specific domains of CCDC93 can rescue the lethal phenotype to identify critical functional regions

  • Expressing human CCDC93 variants in mouse knockout backgrounds to evaluate functional conservation

These approaches provide potential avenues to circumvent the challenges posed by embryonic lethality while still gaining meaningful insights into CCDC93's developmental functions.

How can researchers differentiate between direct and indirect effects of CCDC93 on cellular processes?

Distinguishing direct from indirect effects of CCDC93 on cellular processes requires sophisticated experimental approaches:

• Temporal manipulation strategies:

  • Acute vs. chronic depletion comparisons using inducible knockdown/knockout systems to distinguish primary (direct) from secondary (indirect) effects

  • Time-course experiments following CCDC93 manipulation to establish the sequence of cellular changes

• Molecular interaction verification:

  • Proximity labeling techniques (BioID, APEX) to identify proteins physically associated with CCDC93 in living cells

  • Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) to confirm direct protein-protein interactions in situ

• Domain-specific functional analysis:

  • Structure-function studies using domain deletion or point mutations to map specific interactions to particular CCDC93 regions

  • The varying affinities observed between CCDC93's NN-CH domain and different COMMD proteins demonstrates how such approaches can reveal direct binding specificity

• Reconstitution experiments:

  • In vitro reconstitution of minimal systems (e.g., purified components of the CCC complex) to test whether observed effects can be recapitulated without additional cellular factors

  • The successful demonstration of spontaneous binding between purified CCDC93 and COMMD proteins in vitro exemplifies this approach

• Rescue experiments with specificity controls:

  • Complementation of CCDC93 deficiency with wild-type vs. interaction-deficient mutants

  • Cross-complementation with related proteins (e.g., CCDC22) to test functional specificity

• Pathway analysis with multiple interventions:

  • Comparative transcriptomic/proteomic analyses across different perturbations (e.g., CCDC93 vs. CCDC22 vs. COMMD knockouts)

  • This can help identify effects specific to CCDC93 versus those common to CCC complex disruption