Recombinant Human Cytochrome c oxidase subunit 6C (COX6C)

What is COX6C and what is its role in cellular respiration?

COX6C (Cytochrome c Oxidase subunit 6C) is a component of cytochrome c oxidase (Complex IV), the terminal enzyme in the mitochondrial electron transport chain which drives oxidative phosphorylation. As part of this complex, COX6C contributes to the enzyme that catalyzes the reduction of oxygen to water. The respiratory chain contains three multisubunit complexes: succinate dehydrogenase (Complex II), ubiquinol-cytochrome c oxidoreductase (Complex III), and cytochrome c oxidase (Complex IV), which cooperate to transfer electrons derived from NADH and succinate to molecular oxygen . This process creates an electrochemical gradient across the inner mitochondrial membrane that drives ATP production.

The electron transfer mechanism involves electrons from reduced cytochrome c in the intermembrane space being transferred via the dinuclear copper A center of subunit 2 and heme A of subunit 1 to the active site—a binuclear center formed by heme A3 and copper B. This center reduces molecular oxygen to two water molecules using four electrons from cytochrome c and four protons from the mitochondrial matrix .

How can recombinant COX6C be used in experimental research?

Recombinant Human COX6C protein can be utilized in various experimental applications, particularly:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of COX6C levels in various samples and testing antibody binding specificity.

• Western Blotting (WB): For detection and semi-quantitative analysis of COX6C expression in cell or tissue lysates, allowing comparison between different experimental conditions or disease states .

• Functional Studies: To investigate the role of COX6C in mitochondrial respiration, including overexpression and knockdown experiments to assess its impact on cellular bioenergetics.

• Protein-Protein Interaction Studies: To identify binding partners and understand the structural organization of Complex IV.

• Biomarker Investigation: As potential cancer biomarkers, particularly in pancreatic cancer with KRAS mutations .

What methods are optimal for detecting COX6C expression in pancreatic cancer tissues?

Based on research findings, several complementary methods provide robust detection of COX6C expression in pancreatic cancer tissues:

• Immunohistochemistry (IHC): Particularly effective for tissue microarrays. The protocol involves dewaxing sections, microwave antigen retrieval, endogenous peroxidase blocking, overnight incubation with COX6C antibody (dilution 1:100), followed by secondary antibody incubation for 30 minutes. Detection is performed using DAB (3,3'-diaminobenzidine) and hematoxylin counterstaining .

• Western Blot Analysis: For protein-level quantification in cell lines or tissue homogenates. Proteins are separated on 5-15% SDS-PAGE gels (chosen based on COX6C's molecular weight), transferred to membranes, and detected using specific antibodies .

• Quantitative PCR (qPCR): For mRNA expression analysis using COX6C-specific primers (forward: 5'-ctttgtataagtttcgtgtgg-3' and reverse: 5'-attcatgtgtcatagttcagg-3') .

• PCR Array Analysis: For comparative studies examining multiple mitochondrial genes simultaneously, particularly useful when investigating COX6C in relation to other respiratory chain components .

How does KRAS mutation affect COX6C expression and mitochondrial function?

Research on pancreatic cancer cells has revealed a significant relationship between KRAS mutation and COX6C expression. In a study comparing BxPC-3/KrasG12D mutant cells with wild-type BxPC-3 cells:

• Upregulation of COX6C: KRAS mutation (specifically G12D) significantly increases COX6C expression at both mRNA and protein levels. Among 84 mitochondrial respiration-related genes examined, COX6C showed the highest upregulation in KRAS-mutated cells .

• Enhanced Mitochondrial Function: KRAS-mutated cells displayed:

  • Increased mitochondrial mass

  • Elevated ATP production

  • Higher mitochondrial membrane potential

  • Enhanced COX enzyme activity

• Functional Significance: The relationship appears causal, as siRNA-mediated knockdown of COX6C in KRAS-mutated cells significantly decreased:

  • Cell viability

  • COX enzyme activity

  • ATP production, particularly when mitochondria were supplied with substrates like citrate (60% decrease) and α-ketoglutarate (40% decrease)

This suggests that KRAS mutations may reprogram cancer cell metabolism partly through COX6C upregulation, enhancing oxidative phosphorylation capacity.

What is the cytochrome c oxidase (COX) enzyme activity assay protocol for assessing COX6C function?

For reliable assessment of COX enzyme activity in relation to COX6C function, the following methodological approach is recommended:

• Mitochondrial Isolation:

  • Harvest cells (approximately 10×10^7 cells per condition)

  • Isolate mitochondrial fraction using differential centrifugation

  • Resuspend isolated mitochondria in ice-cold buffer containing 25 mM potassium phosphate, 5 mM MgCl2, 1 mM EDTA, and 0.6 mM lauryl maltoside with protease inhibitors

• Sample Preparation:

  • Subject samples to three freeze-thaw cycles to ensure membrane disruption

  • Determine protein concentration using BCA (bicinchoninic acid) assay

  • Dilute homogenates 10× in assay buffer

• Activity Measurement:

  • Measure enzyme activity spectrophotometrically by following the oxidation of reduced cytochrome c

  • Calculate activity based on the rate of absorbance change

  • Express results as nanomoles of cytochrome c oxidized per minute per milligram of protein

• Controls and Validation:

  • Include appropriate controls (untreated cells, cells with known COX activity levels)

  • Use specific COX inhibitors (e.g., potassium cyanide) to confirm specificity of the assay

How is COX6C expression altered in pancreatic cancer tissues compared to normal tissues?

Immunohistochemical analysis of tissue microarrays containing 30 pairs of pancreatic carcinoma and matched adjacent tissues has revealed significant differences in COX6C expression patterns:

• Expression Pattern:

  • Adjacent normal tissues: Weak cytoplasmic COX6C staining

  • Tumor tissues: Moderate to strong cytoplasmic COX6C staining

• Quantitative Assessment:

  • COX6C immunoreactivity is significantly more intense and diffuse in tumor tissue

  • The difference in expression between tumors and their matched adjacent tissues is statistically significant

• Clinical Implications:

  • Overexpression of COX6C in pancreatic cancer tissues suggests its potential utility as a biomarker

  • The correlation with KRAS mutation status indicates possible involvement in cancer-specific metabolic reprogramming

These findings highlight COX6C as a potentially important molecule in pancreatic cancer biology, warranting further investigation into its role in carcinogenesis and potential as a therapeutic target.

What experimental approach can be used to study the effects of COX6C knockdown on cancer cell metabolism?

To rigorously investigate the effects of COX6C knockdown on cancer cell metabolism, particularly in KRAS-mutated cells, the following comprehensive experimental approach is recommended:

• siRNA-Mediated Knockdown:

  • Design siRNA targeting COX6C (recommended sequence: sense 5'-GGACCACAUUAGGAAGGUUTT-3' and antisense 5'-AACCUUCCUAAUGUGGUCCAG-3')

  • Transfect cells using Lipofectamine RNAiMAX or similar reagent

  • Include negative control siRNA (non-targeting) for all experiments

  • Validate knockdown efficiency by Western blot and qPCR after 48 hours of transfection

• Cell Viability Assessment:

  • Monitor cell proliferation using standard assays (MTT, XTT, or cell counting)

  • Assess colony formation ability in both 2D and 3D culture conditions

• Mitochondrial Function Analysis:

  • Isolate mitochondria from COX6C-knockdown and control cells

  • Measure COX enzyme activity using the spectrophotometric method

  • Quantify ATP production using a bioluminescent ATP determination assay

  • Test mitochondrial substrate utilization by incubating isolated mitochondria with different substrates (10 mM) for 15 minutes at 37°C:

    • Citrate

    • α-ketoglutarate

    • Other TCA cycle intermediates

• Mitochondrial Dynamics Assessment:

  • Evaluate mitochondrial mass using appropriate dyes

  • Measure mitochondrial membrane potential

  • Assess changes in mitochondrial morphology using microscopy

The data from these experiments can be organized in a table format to facilitate comparison between control and COX6C-knockdown conditions:

What are the critical factors for successful expression and purification of recombinant COX6C?

When expressing and purifying recombinant COX6C for research applications, several critical factors should be considered:

• Expression System Selection:

  • Wheat germ cell-free expression systems have proven effective for producing functional human COX6C

  • This system is particularly suitable for membrane-associated proteins like COX6C

  • The expression construct should contain the complete coding sequence (75 amino acids) with appropriate tags for purification

• Protein Solubility Considerations:

  • As a component of a membrane-bound complex, COX6C may present solubility challenges

  • Consider using mild detergents or membrane-mimetic environments during purification

  • Careful optimization of buffer conditions is essential to maintain protein folding and function

• Quality Control Parameters:

  • Verify protein identity by mass spectrometry

  • Confirm structural integrity using circular dichroism or other spectroscopic methods

  • Assess functional activity through interaction studies with other cytochrome c oxidase subunits

• Storage Conditions:

  • Determine optimal storage buffer composition to prevent aggregation or degradation

  • Evaluate protein stability under different temperature conditions (4°C, -20°C, -80°C)

  • Consider the addition of stabilizing agents such as glycerol or specific detergents

How can researchers address experimental variability in COX6C-related ATP production assays?

ATP production assays involving COX6C require careful methodological considerations to ensure reproducibility and reliability:

• Standardized Mitochondrial Isolation:

  • Use a consistent cell number (e.g., 10×10^7 cells) for mitochondrial isolation

  • Maintain identical buffer compositions across experiments

  • Perform isolations at the same time point after treatments

• Substrate Selection and Preparation:

  • Prepare fresh substrate solutions for each experiment

  • Standardize substrate concentrations (e.g., 10 mM)

  • Control the pH of substrate solutions to eliminate variability

• Assay Conditions Optimization:

  • Maintain consistent incubation time (15 minutes) and temperature (37°C)

  • Conduct assays in dark 96-well plates to prevent photodegradation

  • Control for background luminescence in ATP determination assays

• Normalization Strategies:

  • Normalize ATP measurements to protein concentration

  • Consider dual normalization to both protein content and mitochondrial mass

  • Include internal standards in each assay run

• Statistical Approaches for Variability Reduction:

  • Perform triplicate technical replicates for each biological sample

  • Use appropriate statistical tests accounting for experimental variability

  • Report data as mean ± standard deviation from at least three independent experiments

What is the potential of COX6C as a therapeutic target in KRAS-mutated cancers?

Based on current research findings, COX6C shows significant promise as a therapeutic target in KRAS-mutated cancers, particularly pancreatic cancer:

• Rational for Targeting:

  • COX6C is significantly upregulated in KRAS-mutated cancer cells

  • It appears to be functionally important for maintaining enhanced mitochondrial activity and cancer cell viability

  • KRAS mutations are found in approximately 90% of pancreatic cancers but remain largely "undruggable," making downstream effectors like COX6C attractive alternative targets

• Potential Therapeutic Approaches:

  • Small molecule inhibitors specifically targeting COX6C

  • Peptide-based inhibitors disrupting COX6C interactions within the cytochrome c oxidase complex

  • RNA interference strategies for targeted downregulation in cancer cells

  • Antibody-drug conjugates targeting COX6C-overexpressing cells

• Expected Therapeutic Effects:

  • Decreased COX enzyme activity

  • Reduced ATP production from oxidative phosphorylation

  • Metabolic stress in cancer cells highly dependent on mitochondrial function

  • Potential sensitization to existing chemotherapeutic agents

• Considerations for Development:

  • Specificity for cancer cells versus normal cells

  • Delivery strategies to target mitochondria in cancer cells

  • Potential compensatory mechanisms that might emerge

  • Combination strategies with other metabolic inhibitors

How might multi-omics approaches enhance our understanding of COX6C's role in cellular metabolism?

Integrating multiple omics approaches can provide comprehensive insights into COX6C's role in cellular metabolism:

• Genomic Approaches:

  • CRISPR/Cas9 screening to identify synthetic lethal interactions with COX6C in different genetic backgrounds

  • ChIP-seq analysis to identify transcription factors regulating COX6C expression

  • Whole genome sequencing to identify mutations or copy number variations affecting COX6C in patient samples

• Transcriptomic Analysis:

  • RNA-seq to profile global gene expression changes upon COX6C modulation

  • Single-cell RNA-seq to understand cell-specific effects in heterogeneous cancer populations

  • Ribosome profiling to assess translational regulation of COX6C and related genes

• Proteomic Strategies:

  • Interaction proteomics to map the COX6C interactome

  • Phosphoproteomics to identify signaling pathways affected by COX6C modulation

  • Quantitative proteomics to measure changes in mitochondrial protein composition

• Metabolomic Integration:

  • Targeted metabolomics focusing on TCA cycle intermediates and related pathways

  • Stable isotope tracing to track metabolic flux changes upon COX6C manipulation

  • Integration of oxygen consumption measurements with metabolite profiles

• Proposed Multi-omics Workflow:

  • Generate COX6C knockout and overexpression models

  • Perform parallel omics analyses under identical conditions

  • Use computational approaches to integrate datasets

  • Validate key findings with focused biochemical assays

Such comprehensive multi-omics analyses would provide unprecedented insights into how COX6C contributes to metabolic reprogramming in cancer and potentially identify novel therapeutic opportunities beyond direct targeting of COX6C itself.