Recombinant Macaca silenus Cytochrome c oxidase subunit 6C (COX6C)

What are the key characteristics of recombinant Macaca silenus COX6C protein?

Recombinant Macaca silenus COX6C is typically produced as a partial protein in E. coli expression systems with purity exceeding 85% as verified by SDS-PAGE . The protein is characterized by its UniProt accession number Q7YRK2 and can be stored in liquid form for approximately 6 months at -20°C/-80°C, or in lyophilized form for up to 12 months at the same temperature range . For optimal stability and functionality, it is recommended to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol as a cryoprotectant for long-term storage .

How is COX6C expression regulated in cells?

COX6C expression is regulated through multiple pathways:

• PGC-1/ERR signaling pathway: COX6C is induced by the estrogen-associated receptor (ERR), a PGC-1 partner. ERRα target genes contain a conserved ERR responder (ERRE) in their promoters .

• Autoregulatory mechanisms: ERRα binding to ERRE in its own promoter stimulates transcription in a positive autoregulatory loop when activated by PGC-1α .

• MicroRNA regulation: During influenza virus infection, host cells initially boost COX6C mRNA expression by silencing miR-4276. Later, viral control induces miR-4276 expression, which represses COX6C and caspase-9 expression .

• Copy number variations: In certain cancers, such as lung adenocarcinoma, COX6C expression can be enhanced through copy number amplification, particularly of the 8q22.1-22.2 region .

How does COX6C knockdown affect cellular metabolism and what are the downstream consequences?

COX6C knockdown has profound effects on cellular metabolism and function. Research in lung adenocarcinoma cells has demonstrated that COX6C depletion:

• Mitochondrial dysfunction: Causes significant deficiencies in mitochondrial fusion and impairs oxidative phosphorylation .

• ROS accumulation: Triggers reactive oxygen species (ROS) accumulation, activating the AMPK pathway .

• Cell cycle disruption: Induces S-G2/M cell cycle arrest and mitosis deficiency through a mechanism involving:

  • Abnormal spindle formation

  • Chromosome segregation errors

  • Activation of spindle assembly checkpoint

• Apoptotic response: Ultimately leads to cell apoptosis through these cascading events .

These findings suggest that COX6C plays a critical role in maintaining mitochondrial integrity and function, with its disruption triggering a metabolic crisis that impacts multiple cellular processes including cell division and survival.

What is the evolutionary significance of studying COX6C in Macaca silenus compared to other primate species?

Studying COX6C in Macaca silenus carries particular evolutionary significance due to the species' unique phylogenetic positioning within the macaque genus. Recent phylogenomic analyses have revealed that the lion-tailed macaque (M. silenus) and M. leonina are sister species within the silenus group . This challenges previous morphological hypotheses that suggested M. leonina and M. nemestrina were more closely related based on phenotypic appearance and ecological proximity .

Furthermore, studying COX6C across different macaque species provides insight into mitochondrial protein evolution in primates with different evolutionary histories. The silenus group maintains distinct genetic characteristics despite the fascicularis group having originated through ancient hybridization between the sinica and silenus groups approximately 3.45 to 3.56 million years ago . Comparative analysis of COX6C across these lineages may reveal how this essential mitochondrial protein has evolved under different selective pressures and genomic contexts.

What role does COX6C play in viral infection resistance, particularly concerning retroviruses?

COX6C has demonstrated an important role in viral infection resistance, particularly during early stages of infection. During influenza virus exposure, host cells initially boost COX6C mRNA expression by silencing miR-4276, which acts as a defense mechanism. COX6C then inhibits viral replication through activation of caspase-9, driving infected cells into an apoptotic state . This represents an innate cellular defense strategy that eliminates infected cells before the virus can replicate and spread.

Regarding retroviruses specifically, functional experimentation has confirmed that all extant Western silenus species, including Macaca silenus, are susceptible to HIV-1 infection . While the specific role of COX6C in retroviral infections has not been fully elucidated, the connection between mitochondrial function, apoptotic pathways, and viral resistance suggests COX6C could be a target for understanding species-specific susceptibility to retroviruses. Given that COX6C influences apoptotic pathways, it may contribute to the cellular response to retroviral infection, potentially affecting viral replication dynamics and cell survival.

What are the optimal methods for expressing and purifying recombinant Macaca silenus COX6C?

Expression System Selection:
E. coli is the preferred expression system for recombinant Macaca silenus COX6C due to its cost-effectiveness and high yield potential . For researchers requiring post-translational modifications, mammalian or insect cell expression systems may be more appropriate, though with lower yields.

Purification Protocol:

• Cell Lysis: Optimal lysis using sonication or French press in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors.

• Affinity Chromatography: If expressing with a tag (His or GST), use corresponding affinity resin.

• Size Exclusion Chromatography: Further purification using Superdex 75 or equivalent.

• Yield Assessment: Typical yield ranges from 2-5 mg/L of culture with >85% purity as determined by SDS-PAGE .

• Quality Control: Confirm identity by mass spectrometry and functional activity through specific enzymatic assays.

Storage Recommendations:

• Store as aliquots in buffer containing 20-50% glycerol at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

What experimental approaches are most effective for studying COX6C function in mitochondrial respiration?

Cellular Models:

• Knockdown/Knockout Systems: siRNA, shRNA, or CRISPR-Cas9 approaches to reduce or eliminate COX6C expression .

• Overexpression Systems: Transfection with COX6C expression vectors to examine gain-of-function effects.

Functional Assays:

• Oxygen Consumption Rate (OCR) Measurement: Using Seahorse XF analyzers to quantify mitochondrial respiration rates in intact cells.

• Cytochrome c Oxidase Activity Assay: Spectrophotometric measurement of cytochrome c oxidation rate.

• ATP Production Assay: Luminescence-based quantification of cellular ATP levels.

• Mitochondrial Membrane Potential Assessment: Using fluorescent dyes like JC-1, TMRE, or Rhodamine 123.

Microscopy Techniques:

• Confocal Microscopy: To visualize mitochondrial network morphology using MitoTracker dyes.

• Super-resolution Microscopy: For detailed examination of COX complex assembly.

• Transmission Electron Microscopy: For ultrastructural analysis of mitochondrial cristae morphology.

Molecular Interaction Studies:

• Co-immunoprecipitation: To identify protein-protein interactions within the COX complex.

• Blue Native PAGE: For analysis of intact respiratory complexes and supercomplexes.

How can researchers effectively measure changes in COX6C expression in response to various cellular stressors?

RNA Expression Analysis:

• RT-qPCR: For quantitative measurement of COX6C mRNA expression with reference genes such as GAPDH or β-actin for normalization.

• RNA-Seq: For genome-wide expression profiling to contextualize COX6C changes within broader transcriptional networks.

Protein Expression Analysis:

• Western Blotting: Using specific anti-COX6C antibodies with appropriate loading controls (β-actin, GAPDH, or other mitochondrial proteins).

• Immunocytochemistry: For spatial localization of COX6C within cells under different stress conditions.

• Proteomics: Mass spectrometry-based approaches for unbiased quantification of protein abundance changes.

Stress Induction Protocols:

• Hypoxia: Culture cells at 1-3% O₂ for varying durations to examine adaptive responses .

• Oxidative Stress: Treatment with H₂O₂, paraquat, or rotenone.

• Metabolic Stress: Glucose deprivation, fatty acid overload, or galactose media to force OXPHOS-dependent ATP production.

• Viral Infection Models: Using influenza virus or HIV-1 to study infection-specific responses .

Temporal Dynamics:

• Time-course Experiments: Critical for capturing both early regulatory responses and later adaptive changes.

• Pulse-Chase Labeling: To distinguish between changes in synthesis versus degradation rates.

How does the role of COX6C differ between normal cellular function and cancer progression?

In normal cellular function, COX6C serves as a critical structural subunit of the cytochrome c oxidase complex, facilitating efficient electron transfer and ATP production through oxidative phosphorylation. It contributes to maintaining mitochondrial integrity and function under physiological conditions.

In cancer contexts, COX6C exhibits notable differences in expression and function:

The paradoxical role of COX6C in cancer cells relates to the Warburg effect, where cancer cells often rely on glycolysis despite adequate oxygen availability. Despite this metabolic shift, many cancer cells maintain or upregulate components of the respiratory chain, including COX6C, suggesting these components may serve functions beyond their canonical roles in OXPHOS. COX6C overexpression may contribute to cancer cell survival by facilitating mitochondrial dynamics, regulating apoptotic thresholds, or contributing to redox homeostasis .

What are the key differences between COX6C from Macaca silenus and human COX6C in terms of structure, function, and experimental applications?

Structural Comparison:

Functional Comparison:
Both proteins serve similar fundamental roles in mitochondrial respiration, but species-specific differences may exist in regulation and protein-protein interactions. The high sequence conservation suggests functional similarity, making Macaca silenus COX6C a valuable model for understanding human COX6C function.

Experimental Applications:

• Cross-reactivity: Antibodies against human COX6C often cross-react with macaque COX6C, facilitating comparative studies.

• Animal Models: Studying COX6C in macaques provides insights applicable to human disease within a more closely related primate model.

• Evolutionary Studies: Comparing COX6C across primates helps trace the evolution of mitochondrial function in our lineage.

• Viral Susceptibility Research: The susceptibility of Macaca silenus to HIV-1 makes it valuable for studying COX6C's role in retroviral infection .

What pathological conditions involve COX6C dysregulation and what are the underlying mechanisms?

COX6C dysregulation has been implicated in multiple pathological conditions through various mechanisms:

Underlying Mechanisms:

• Genomic Amplification: In lung adenocarcinoma, copy number amplification of 8q22.2 drives COX6C overexpression .

• Transcriptional Regulation: Dysregulation of PGC-1/ERR signaling pathway affects COX6C expression .

• Post-transcriptional Control: microRNAs like miR-4276 regulate COX6C levels during viral infections .

• Metabolic Feedback Loops: Changes in cellular metabolism alter COX6C expression and activity.

• Oxidative Stress: ROS accumulation influences COX6C function and can trigger pathological cascades, including mitotic defects and apoptosis .

What are the most promising approaches for targeting COX6C in cancer therapy?

Given the involvement of COX6C in cancer progression, several therapeutic approaches warrant investigation:

• Small Molecule Inhibitors: Developing specific inhibitors targeting COX6C or its interactions within the respiratory complex.

• RNA Interference Technologies: siRNA or antisense oligonucleotides targeting COX6C mRNA, particularly in cancers with COX6C overexpression .

• PROTAC (Proteolysis-Targeting Chimeras): Molecules designed to target COX6C for ubiquitin-proteasome degradation.

• Combination Therapies: Pairing COX6C-targeting approaches with conventional chemotherapies or radiation.

• Biomarker Development: Using COX6C expression levels for patient stratification and treatment selection, especially in lung adenocarcinoma patients with 8q22.2 amplification .

Research indicates that COX6C knockdown in lung adenocarcinoma cells induces mitochondrial dysfunction, ROS accumulation, cell cycle arrest, and apoptosis through AMPK pathway activation . This mechanistic understanding provides a foundation for therapeutic development, suggesting that COX6C inhibition could trigger selective cancer cell death while potentially sparing normal cells with lower dependency on COX6C function.

How might comparative studies of COX6C across macaque species inform our understanding of mitochondrial evolution and disease susceptibility?

Comparative studies of COX6C across macaque species represent a valuable approach to understanding both evolutionary biology and disease mechanisms:

• Evolutionary Insights:

  • Tracing COX6C sequence and functional changes across macaque lineages, particularly between the hybrid-origin fascicularis group and the sinica and silenus groups .

  • Examining selection pressures on COX6C in different ecological niches and environmental conditions.

  • Understanding how hybridization events have shaped mitochondrial protein evolution.

• Disease Susceptibility:

  • Comparing COX6C function in species with different susceptibilities to diseases like viral infections.

  • Testing whether specific COX6C variants correlate with resistance to metabolic disorders or cancer.

  • The finding that all Western silenus species are susceptible to HIV-1 infection provides a framework for investigating whether mitochondrial proteins like COX6C play roles in viral susceptibility.

• Methodological Approaches:

  • Genomic and transcriptomic comparisons across species.

  • Functional testing of COX6C variants in controlled cellular systems.

  • Development of species-specific antibodies and assays to compare COX6C expression and activity.

Such comparative studies could reveal critical functional domains within COX6C and identify naturally occurring variations that confer advantages in mitochondrial function or disease resistance, ultimately informing therapeutic strategies for human diseases involving mitochondrial dysfunction.