Recombinant Mouse Coiled-coil domain-containing protein 109B (Ccdc109b)

What is the molecular structure and subcellular localization of CCDC109B?

CCDC109B contains a coiled-coil domain structural motif and two predicted transmembrane domains. It was first identified as a paralogue of the Mitochondrial Calcium Uniporter (MCU) . The protein primarily localizes to the inner mitochondrial membrane (IMM) where it plays a role in facilitating Ca²⁺ flux. Immunofluorescence studies have confirmed its expression pattern, showing differential distribution between normal human astrocytes and glioma cells .

Methodologically, researchers can visualize CCDC109B distribution using confocal microscopy with cells fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with primary antibody against CCDC109B (1:100) followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG (1:800) .

How does CCDC109B function in normal cellular processes?

In normal cells, CCDC109B acts as a dominant negative mediator of MCU, attenuating mitochondrial calcium increases evoked by agonist stimulation. This regulation of calcium homeostasis is crucial for mitochondrial function and cellular metabolism . Experimental approaches to study this function include calcium flux assays and mitochondrial function assessments.

The protein's normal physiological roles include:

• Regulation of mitochondrial Ca²⁺ levels

• Maintenance of mitochondrial membrane potential

• Potential influence on cellular energy metabolism

• Modulation of cellular response to environmental stressors

What experimental techniques are most reliable for measuring CCDC109B expression levels?

For quantifying CCDC109B expression, multiple complementary approaches should be employed:

• Western blot analysis: Protein isolation using RIPA buffer containing protein inhibitor cocktail, followed by SDS-PAGE separation and transfer to PVDF membranes. Primary antibody against CCDC109B at 1:500 dilution provides reliable results .

• qRT-PCR: For mRNA quantification, with appropriate housekeeping gene controls.

• Immunohistochemistry (IHC): For tissue sections, allowing assessment of expression patterns across different regions of tissue samples .

• Immunofluorescence (IF): For subcellular localization studies, particularly valuable for co-localization experiments .

For validation, cross-referencing results with public databases (TCGA, CGGA, and GEO) is recommended for comparative analysis.

What are the most effective approaches for CCDC109B knockdown in experimental systems?

Based on experimental validation, lentiviral-mediated shRNA delivery provides consistent and stable CCDC109B knockdown. Three different shRNAs targeting CCDC109B have been tested, with sh-CCDC109B-1 demonstrating nearly complete protein depletion, making it optimal for functional studies .

Experimental protocol details:

• Design multiple shRNA sequences targeting different regions of CCDC109B mRNA

• Package into lentiviral vectors for stable integration

• Verify knockdown efficiency by qRT-PCR and western blot analysis

• For in vivo studies, establish stable cell lines before implantation

Alternative approaches include siRNA for transient knockdown, particularly useful in hypoxia-related experiments where HIF1α and CCDC109B relationships are being investigated .

How can researchers effectively model hypoxia-induced changes in CCDC109B expression?

Hypoxia significantly affects CCDC109B expression, making it an important experimental variable. The recommended protocol includes:

• Culture cells in a hypoxic chamber (1% O₂, 5% CO₂, 94% N₂) for 24-48 hours

• For pharmacological hypoxia induction, treat cells with cobalt chloride (CoCl₂, 100-200 μM)

• Verify hypoxic conditions by measuring HIF1α expression

• Compare CCDC109B expression between normoxic and hypoxic conditions using western blot and qRT-PCR

For in vivo studies, administration of HIF1α inhibitors such as digoxin (2 mg/kg daily by intraperitoneal injection) can be used to modulate the hypoxic response and subsequently CCDC109B expression .

What cell and animal models provide the most relevant systems for studying CCDC109B in glioma?

Cell models:

• U87MG and U251 human glioma cell lines have been validated for CCDC109B functional studies

• These cell lines demonstrate measurable CCDC109B expression and respond to both genetic manipulation and hypoxic conditions

• Normal human astrocytes serve as appropriate controls

Animal models:

How does CCDC109B expression correlate with glioma grade and patient prognosis?

CCDC109B expression shows a strong correlation with glioma grade and patient outcomes:

Expression by tumor grade:

• High-grade gliomas (WHO III-IV): 59.2% (29/49) high expression

• Low-grade gliomas (WHO I-II): 10.5% (2/19) high expression

• Normal brain tissue: Minimal expression

Prognosis correlation:

What molecular mechanisms underlie CCDC109B's role in glioma cell invasion and migration?

CCDC109B influences glioma invasion and migration through multiple mechanisms:

• Regulation of matrix metalloproteinases: CCDC109B knockdown reduces MMP2 and MMP9 expression, crucial mediators of extracellular matrix degradation during invasion .

• Hypoxia-mediated invasion: CCDC109B is a critical factor in mediating HIF1α-induced glioma cell migration and invasion. Knockdown of CCDC109B significantly attenuates hypoxia-enhanced invasion and migration .

• Mesenchymal phenotype association: CCDC109B expression is significantly elevated in the mesenchymal molecular subtype of glioma, which is characterized by enhanced invasive properties .

Experimental Transwell migration and invasion assays have quantitatively demonstrated that CCDC109B knockdown reduces the number of migrating/invading cells by approximately 50-60% in both U87MG and U251 cell lines .

What is the relationship between hypoxia, HIF1α, and CCDC109B expression?

The relationship between hypoxia and CCDC109B has been well-established:

• CCDC109B expression is significantly elevated in necrotic/hypoxic areas of glioma tissue samples .

• In vitro hypoxia (1% O₂) induces CCDC109B expression at both mRNA and protein levels in a time-dependent manner, with peak expression after 24 hours of hypoxic exposure .

• HIF1α appears to be a direct transcriptional regulator of CCDC109B:

  • siRNA-mediated knockdown of HIF1α reduces CCDC109B expression

  • Pharmacological inhibition of HIF1α using specific inhibitors (YC-1 or digoxin) decreases CCDC109B expression both in vitro and in vivo

  • In vivo digoxin treatment (2 mg/kg) significantly reduces both tumor size and CCDC109B expression in xenograft models

This regulatory relationship suggests that CCDC109B may be an important downstream effector of hypoxia-induced changes in glioma progression.

How might CCDC109B serve as a therapeutic target in glioma treatment?

CCDC109B presents several attributes of a promising therapeutic target:

The research suggests that CCDC109B targeting may be particularly valuable in hypoxic tumors, which are often more resistant to conventional therapies.

What research gaps remain in understanding CCDC109B's role in brain tumors?

Despite significant progress, several key questions remain:

• Precise molecular mechanisms: How does CCDC109B influence mitochondrial calcium handling in glioma cells, and how does this connect to proliferation and invasion?

• Interaction partners: The complete interactome of CCDC109B in glioma cells remains to be elucidated.

• Genetic and epigenetic regulation: Beyond HIF1α, what other factors regulate CCDC109B expression?

• Relationship to treatment resistance: Preliminary data suggest CCDC109B may contribute to temozolomide resistance, but mechanisms remain unclear .

• Therapeutic targeting: No specific CCDC109B inhibitors have been developed or tested.

Future research should employ multi-omics approaches, detailed structure-function analysis, and development of more sophisticated in vivo models to address these questions.

How does CCDC109B expression vary across glioma molecular subtypes?

CCDC109B shows distinct expression patterns across different molecular subtypes of glioma:

This pattern has been consistently observed across multiple datasets (TCGA, CGGA, GSE4271) . The association with the mesenchymal subtype is particularly interesting as this subtype is typically associated with more aggressive behavior and poorer outcomes.

The elevated expression in mesenchymal gliomas suggests that CCDC109B may play a role in the mesenchymal transition process or in maintaining mesenchymal characteristics, further supporting its potential role in invasion and migration.

What are the common challenges in CCDC109B protein detection and quantification?

Researchers commonly encounter several technical challenges when studying CCDC109B:

• Antibody specificity: Due to structural similarities with other coiled-coil domain proteins, antibody cross-reactivity can occur. Validation using positive and negative controls is essential.

• Mitochondrial protein extraction: As CCDC109B localizes to mitochondria, standard protein extraction protocols may not be optimal. Specialized mitochondrial isolation procedures may improve detection.

• Protein stability: Attention to sample handling and processing time is critical for accurate quantification.

Best practices include:

• Using standardized protein extraction protocols with RIPA buffer containing protease inhibitors

• Processing samples rapidly at 4°C

• Including appropriate controls (knockdown samples as negative controls)

• Validating antibodies using multiple detection methods

How can researchers effectively design functional studies to investigate CCDC109B's role in specific cellular processes?

For comprehensive functional characterization of CCDC109B, consider these methodological approaches:

• Gene manipulation strategies:

  • Use multiple shRNA/siRNA sequences to control for off-target effects

  • Complement knockdown studies with rescue experiments

  • Consider inducible systems for temporal control of expression

• Comprehensive functional assays:

  • Proliferation: EdU incorporation and colony formation assays

  • Migration/invasion: Transwell assays with/without matrigel coating

  • Calcium signaling: Fluorescent calcium indicators to measure mitochondrial calcium flux

  • In vivo studies: Both orthotopic and subcutaneous xenograft models

• Contextual factors to consider:

  • Oxygen levels: Compare normoxic vs. hypoxic conditions

  • Growth factors: Examine interactions with tumor microenvironment signals

  • Drug treatments: Assess impact on treatment response

• Data validation:

  • Use multiple cell lines (minimum of two distinct glioma lines)

  • Validate key findings in patient-derived primary cultures

  • Correlate in vitro findings with patient data from public databases