Recombinant Mouse Coiled-coil domain-containing protein 68 (Ccdc68)

What is Mouse Coiled-coil Domain-containing Protein 68 (Ccdc68)?

Mouse CCDC68 is a 38.0 kDa protein that functions as a centriolar protein required for centriole subdistal appendage assembly and microtubule anchoring in interphase cells. It cooperates with CCDC120 and subdistal appendage components ODF2, NIN, and CEP170 for hierarchical subdistal appendage assembly . The protein is encoded by the Ccdc68 gene and contains coiled-coil domains that facilitate protein-protein interactions essential for its structural and functional roles.

What expression systems are recommended for producing recombinant Mouse CCDC68?

For recombinant Mouse CCDC68 production, mammalian expression systems, particularly HEK-293 cells, have proven effective for generating properly folded and functionally active protein . Alternative systems include cell-free protein synthesis (CFPS), which can yield proteins with 70-80% purity as determined by SDS-PAGE, Western Blot, and analytical SEC (HPLC) . When selecting an expression system, researchers should consider:

• Post-translational modifications required for function

• Experimental needs for purity levels

• Tag selection (His, Strep, or Myc-DYKDDDDK tags are commonly used)

• Downstream applications (structural studies, functional assays, or antibody production)

What methods are most effective for detecting CCDC68 expression in tissue samples?

Multiple complementary methods can effectively detect CCDC68 expression:

• Immunohistochemistry (IHC): Effective for tissue localization using specific antibodies against CCDC68 (e.g., PA5-61687; Invitrogen). The standard protocol involves:

  • Formalin-fixed paraffin-embedded tissue sectioning

  • Antigen retrieval

  • Primary antibody incubation

  • Detection with appropriate visualization systems

  • Quantification by calculating the integrated optical density (IOD) of stained areas, with at least five images per specimen recommended for accurate assessment

• Quantitative RT-PCR: For mRNA expression analysis using primers:

  • Forward: 5′-TCTGCCTTGTATGAGTCTACGTCC-3′

  • Reverse: 5′-AGGATCCATTTCAGAATCAGAGCC-3′

• Western Blotting: For protein level detection, using:

  • Lysis buffer containing 10 mM Tris-HCl, 1 mM Na3VO4, and 1% SDS (pH 7.4)

  • SDS-PAGE separation followed by transfer to PVDF membranes

  • Probing with anti-CCDC68 antibodies and appropriate secondary antibodies

  • Detection using ECF western blotting systems

How can researchers assess the impact of CCDC68 on cell proliferation?

Multiple complementary assays can be employed to comprehensively evaluate CCDC68's effect on cell proliferation:

• Cell Viability Assay: Using Cell Titer-Glo Luminescent Cell Viability Assay kit (G7572; Promega):

  • Seed 2000 cells per well in 96-well plates

  • Synchronize cells by exposure to 0.1% FBS medium for 12 hours

  • Culture in complete medium for designated time periods

  • Measure cell viability at days 1, 3, and 5

  • Calculate proliferation rate as the relative absorbance of cells at days 3 and 5 versus day 1

• Anchorage-independent Growth Assay:

  • Prepare 6-well plates with 0.5% agar in Basal Medium Eagle (BME) containing 10% FBS as the bottom layer

  • Suspend 1 × 10^4 cells in 1 mL of 0.33% agar in 10% FBS-BME and seed on the bottom layer

  • Culture for 2-4 weeks in a 5% CO2 incubator

  • Count and measure the size of cell colonies under a microscope

• Cell Cycle Analysis by Flow Cytometry:

  • Collect and fix cells with 70% ethanol at 4°C overnight

  • Wash and stain with propidium iodide and RNase A (9:1)

  • Analyze using flow cytometry and appropriate software for cell cycle distribution

Studies have shown that CCDC68 overexpression significantly decreases both monolayer growth and anchorage-independent growth of colorectal cancer cells compared to control vector transfectants .

What is known about CCDC68's role in the RXRα/ITCH/CDK4 signaling axis?

CCDC68 functions as a tumor suppressor by regulating the RXRα/ITCH/CDK4 signaling axis:

• Mechanism: CCDC68 suppresses colorectal cancer cell proliferation by promoting ITCH transcription, which is mediated by upregulation of the transcription factor RXRα .

• Downstream Effects: This signaling cascade alters cyclin-dependent kinase 4 (CDK4) protein degradation, with CCDC68 overexpression leading to decreased CDK4 protein levels .

• Functional Validation: When CDK4 is ectopically expressed in CCDC68-overexpressing cells, it reverses the growth-inhibitory effects of CCDC68, confirming CDK4 as a downstream effector of CCDC68-mediated growth suppression .

• Cell Cycle Impact: CCDC68 induces G0/G1 growth arrest, which can be reversed by CDK4 overexpression, indicating that CDK4 downregulation is a key mechanism by which CCDC68 inhibits cell proliferation .

How can researchers generate stable CCDC68-expressing cell lines for long-term studies?

For establishing stable CCDC68-expressing cell lines:

• Vector Selection:

  • Choose appropriate expression vectors with selection markers (e.g., neomycin, puromycin resistance)

  • Consider tag addition (His, HA, or GFP tags) for detection and functional studies

  • Ensure the vector contains appropriate promoters for sustained expression

• Transfection Methodology:

  • For colorectal cancer cell lines like HCT116 and RKO, lipid-based transfection reagents have proven effective

  • Optimize transfection conditions for your specific cell type

• Selection Process:

  • Begin selection 24-48 hours post-transfection with appropriate antibiotics

  • Maintain selection pressure for 2-3 weeks to eliminate non-transfected cells

  • Isolate single colonies for expansion of clonal populations

• Validation of Expression:

  • Confirm stable expression by western blotting

  • Assess protein functionality through appropriate assays

  • Monitor expression stability over multiple passages

Researchers have successfully established stable CCDC68-expressing HCT116 and RKO cell lines, which demonstrated consistent inhibition of colorectal cancer cell growth in multiple experimental settings .

What experimental approaches can determine CCDC68's effect on protein degradation pathways?

To study CCDC68's impact on protein degradation pathways, particularly for targets like CDK4:

• Protein Stability Assay:

  • Culture stably transfected cells (e.g., HCT116 vector vs. HCT116 CCDC68) to 50-60% confluence

  • Synchronize cells by starvation in 0.1% FBS medium for 12 hours

  • Culture in 10% FBS medium for 12 hours

  • Treat with proteasome inhibitor MG132 (10 μM) for 5 hours

  • Expose to protein synthesis inhibitor cycloheximide (CHX, 50 μg/mL) for varying durations (0, 3, 6, and 12 hours)

  • Analyze protein levels by western blotting at each time point

• Ubiquitination Analysis:

  • Identify potential E3 ligases using databases like UbiBrowser

  • Perform co-immunoprecipitation to detect ubiquitinated forms of the target protein

  • Compare ubiquitination levels between control and CCDC68-overexpressing cells

• Promoter Activity Assay:

  • For studying transcriptional regulation (e.g., ITCH promoter activity):

  • Co-transfect cells with promoter-driven luciferase reporter and pRL-TK

  • After 24 hours, lyse cells and measure luciferase activity

  • Normalize to TK activity for accurate comparison

How does CCDC68 contribute to centriole subdistal appendage assembly?

CCDC68 plays a critical role in centriole subdistal appendage assembly and microtubule anchoring:

• Protein Interactions: CCDC68 cooperates with CCDC120 and works with subdistal appendage components ODF2, NIN, and CEP170 in a hierarchical assembly process .

• Experimental Approaches to Study This Function:

  • Immunofluorescence microscopy: To visualize co-localization with other centriolar proteins

  • Proximity ligation assays: To detect protein-protein interactions at centrioles

  • Super-resolution microscopy: To map the precise localization within centriolar structures

  • Knockout/knockdown studies: To assess the impact of CCDC68 depletion on centriole structure and function

• Functional Consequences: Proper CCDC68 function is essential for microtubule anchoring in interphase cells, which affects cellular processes including cell division, intracellular transport, and maintenance of cell shape and polarity .

What are common challenges in CCDC68 protein purification and storage?

When working with recombinant CCDC68 protein:

• Purification Challenges:

  • Protein solubility issues may arise due to coiled-coil domains

  • Optimize lysis buffers to enhance solubility

  • Consider using fusion tags (e.g., His, Strep) that facilitate purification while maintaining protein function

  • Purification results can be validated by Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC (HPLC)

• Storage Recommendations:

  • Store purified CCDC68 at -80°C for long-term preservation

  • Avoid repeated freeze-thaw cycles to maintain protein integrity

  • Consider adding stabilizing agents such as glycerol or specific buffer components as needed

  • Aliquot the protein to minimize freeze-thaw cycles when using in experiments

What controls should be included when studying CCDC68's tumor suppressive effects?

For robust experimental design when investigating CCDC68's tumor suppressive functions:

• Essential Controls:

  • Vector-only controls paired with CCDC68-expressing cells

  • Wild-type vs. mutant CCDC68 to identify critical functional domains

  • Rescue experiments (e.g., CDK4 overexpression in CCDC68-expressing cells) to validate downstream effectors

• In Vivo Controls:

  • For xenograft models, include both vector control and CCDC68-expressing cells

  • Monitor tumor growth over time with regular measurements

  • Confirm CCDC68 expression in resulting tumors to verify stability of expression

• Statistical Analysis:

  • Use appropriate statistical methods (e.g., Student's t-test for comparing two groups)

  • Present data as mean ± standard deviation

  • Consider p < 0.05 as statistically significant

  • Use software like GraphPad Prism for analysis and visualization