Recombinant Nycticebus coucang Cytochrome c oxidase subunit 6C (COX6C)

What is Nycticebus coucang Cytochrome c oxidase subunit 6C (COX6C)?

Nycticebus coucang Cytochrome c oxidase subunit 6C (COX6C) is a nuclear-encoded structural subunit of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain. This protein is derived from the slow loris (Nycticebus coucang) and functions as part of the complex that catalyzes electron transfer from reduced cytochrome c to oxygen in the inner mitochondrial membrane. Cytochrome c oxidase is a heteromeric complex consisting of 3 catalytic subunits encoded by mitochondrial genes and multiple structural subunits encoded by nuclear genes, with COX6C being one of the nuclear-encoded components potentially involved in the regulation and assembly of the complex .

The COX6C protein is also known by alternative names including cytochrome c oxidase polypeptide VIc. It represents an important component for studying mitochondrial function and has applications in various research contexts including cancer biology, where homologous proteins have been shown to be upregulated in certain cancer types .

What are the structural characteristics of Nycticebus coucang COX6C?

Nycticebus coucang COX6C is a small protein with the following structural characteristics:

• Full mature protein length: 74 amino acids (positions 2-75)

• Molecular weight: Approximately 8.5 kDa (based on amino acid composition)

• Secondary structure: Contains hydrophobic regions consistent with membrane association in the mitochondrial inner membrane

• Post-translational modifications: May undergo processing to remove the signal peptide

The protein sequence suggests a structure that facilitates its integration into the cytochrome c oxidase complex within the mitochondrial membrane. The mature protein contains regions that allow for proper folding and interaction with other subunits of the complex to maintain functional integrity of the enzyme .

How is recombinant Nycticebus coucang COX6C typically produced?

Recombinant Nycticebus coucang COX6C is typically produced using bacterial expression systems, primarily E. coli. The production process involves the following key steps:

• Gene synthesis or cloning of the COX6C coding sequence from Nycticebus coucang

• Insertion into an appropriate expression vector with a histidine tag (commonly N-terminal His tag)

• Transformation into competent E. coli cells

• Induction of protein expression (typically using IPTG for lac promoter-based systems)

• Cell lysis and protein extraction

• Purification using affinity chromatography (His-tag purification)

• Quality control assessment (SDS-PAGE, Western blot)

• Lyophilization for stable storage

The resulting recombinant protein typically achieves purity levels greater than 90% as determined by SDS-PAGE analysis . This method allows for the production of consistent, high-quality protein suitable for various research applications, though researchers should be aware that bacterial expression may lack some post-translational modifications present in the native protein.

How should recombinant Nycticebus coucang COX6C be stored and handled?

Proper storage and handling of recombinant Nycticebus coucang COX6C is essential for maintaining its stability and biological activity. Recommended protocols include:

Storage conditions:

• Long-term storage: Store at -20°C to -80°C

• For extended storage, aliquoting is necessary to avoid repeated freeze-thaw cycles

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

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage

Handling precautions:

• Avoid repeated freeze-thaw cycles as they may lead to protein denaturation and loss of activity

• When working with the protein, maintain cold chain conditions whenever possible

• For experimental use, prepare fresh working dilutions on the day of the experiment

What experimental models utilize Nycticebus coucang COX6C?

While research specifically with Nycticebus coucang COX6C is limited in the available literature, the protein has applications in several experimental contexts:

• Comparative biochemistry studies: Researchers use COX6C from various species, including Nycticebus coucang, to study evolutionary conservation of mitochondrial proteins.

• Mitochondrial function assays: The recombinant protein can be used in reconstitution experiments to study cytochrome c oxidase assembly and function.

• Cancer research models: Based on studies with human COX6C, which has shown upregulation in certain cancer types, the Nycticebus coucang ortholog may be useful in comparative oncology research.

• Protein-protein interaction studies: The His-tagged recombinant protein enables pull-down experiments to identify binding partners.

In one documented case, human COX6C has been studied in pancreatic cancer cell lines (BxPC-3) with Kras G12D mutations, where it showed significant upregulation. Similar experimental approaches could be applied using the Nycticebus coucang variant for comparative studies .

How does COX6C function in the mitochondrial respiratory chain?

COX6C functions as a structural subunit of cytochrome c oxidase (Complex IV), which is the terminal enzyme of the mitochondrial respiratory chain. Its key functional roles include:

• Assembly and stability: COX6C contributes to the proper assembly and structural stability of the cytochrome c oxidase complex.

• Regulatory function: While not directly involved in electron transfer (which is primarily mediated by the mitochondrially-encoded catalytic subunits), COX6C likely plays a regulatory role in modulating enzyme activity.

• Complex organization: It helps maintain the quaternary structure of the cytochrome c oxidase complex, enabling efficient electron transfer from reduced cytochrome c to oxygen.

• Energy coupling: By supporting the function of cytochrome c oxidase, COX6C indirectly contributes to the generation of the proton gradient used for ATP synthesis.

Research has demonstrated that alterations in COX6C expression can significantly impact cytochrome c oxidase activity. For instance, in pancreatic cancer cells with Kras G12D mutation, COX6C upregulation corresponded with a 35% increase in cytochrome c oxidase enzymatic activity compared to wild-type cells .

What is the role of COX6C in cancer research, particularly pancreatic cancer?

COX6C has emerged as a significant protein of interest in cancer research, with particular relevance to pancreatic cancer:

• Upregulation in Kras-mutated cells: Studies have demonstrated that COX6C is significantly upregulated in pancreatic cancer cells harboring the Kras G12D mutation. Expression analysis revealed an 18.2-fold elevation in mRNA levels and a 4.6-fold increase in protein expression in BxPC-3/Kras G12D cells compared to wild-type BxPC-3 cells .

• Correlation with enzymatic activity: The overexpression of COX6C correlates with increased cytochrome c oxidase activity (approximately 35% higher) in Kras G12D mutant cells compared to wild-type cells .

• Tissue expression patterns: Immunohistochemistry analysis using tissue microarrays has shown differential expression patterns of COX6C between pancreatic carcinoma and adjacent normal tissues, with weak cytoplasmic staining observed in adjacent tissues compared to cancer samples .

• Functional significance: Knockdown experiments using siRNA targeting COX6C have demonstrated changes in:

  • Cell viability

  • Cytochrome c oxidase activity

  • ATP production

These findings suggest that COX6C may represent a potential new protein highly driven by Kras G12D mutation in pancreatic cancer, making it a valuable target for both diagnostic and therapeutic research .

How can COX6C activity be measured in experimental settings?

Measuring COX6C activity in experimental settings typically involves assessing the function of the entire cytochrome c oxidase complex rather than the individual subunit. Key methodological approaches include:

• Cytochrome c oxidase enzymatic activity assay:

  • Isolation of mitochondria from cells or tissues

  • Measurement of the rate of cytochrome c oxidation spectrophotometrically

  • Quantification of activity as the decrease in absorbance at 550 nm over time

  • Normalization to protein concentration

• Oxygen consumption measurements:

  • Use of oxygen electrodes or plate-based respirometry systems

  • Measurement of oxygen consumption rates in isolated mitochondria or intact cells

  • Addition of specific substrates and inhibitors to isolate Complex IV activity

• ATP production assays:

  • Measurement of ATP levels using bioluminescent assays following treatments

  • In studies with Kras G12D cells, ATP production was assessed after incubation with specific substrates like citrate (Cit) and α-ketoglutarate (AKG) for 15 minutes at 37°C

• Expression analysis:

  • Quantitative PCR to measure mRNA levels

  • Western blotting to quantify protein expression, standardized to loading controls like GAPDH

  • Immunohistochemistry for tissue expression patterns

What are the challenges in working with recombinant COX6C proteins?

Researchers face several challenges when working with recombinant COX6C proteins:

• Protein stability issues:

  • COX6C is sensitive to freeze-thaw cycles, requiring careful aliquoting and storage

  • The recombinant protein may have different stability characteristics compared to the native protein within the cytochrome c oxidase complex

• Functional reconstitution:

  • As a subunit of a multi-protein complex, isolated COX6C may not exhibit independent enzymatic activity

  • Reconstitution into functional cytochrome c oxidase complexes presents technical challenges

• Expression system limitations:

  • E. coli-expressed COX6C lacks post-translational modifications that may be present in the native protein

  • The presence of tags (such as His-tags) may interfere with certain functional assays or structural studies

• Species differences:

  • While studying Nycticebus coucang COX6C offers valuable comparative insights, extrapolation to human systems requires careful validation

  • Sequence variations between species may lead to functional differences

• Methodological considerations:

  • Maintaining the native conformation during experimental procedures can be difficult

  • Proper reconstitution from lyophilized form requires strict adherence to protocols to prevent protein aggregation or denaturation

What reconstitution protocols are recommended for lyophilized Nycticebus coucang COX6C?

The reconstitution of lyophilized Nycticebus coucang COX6C requires careful attention to detail to maintain protein stability and activity. The recommended protocol includes:

• Initial preparation:

  • Bring the vial to room temperature

  • Briefly centrifuge the vial prior to opening to bring the contents to the bottom and prevent loss of material

• Reconstitution procedure:

  • Add deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

  • Gently mix by inverting or slow pipetting to avoid introducing bubbles or causing protein denaturation

  • Do not vortex the solution

• Stabilization:

  • Add glycerol to a final concentration of 5-50% (optimally 50%)

  • This helps maintain protein stability during storage

  • Mix gently until homogeneous

• Storage of reconstituted protein:

  • Aliquot the reconstituted protein into appropriate volumes for single use

  • Store aliquots at -20°C/-80°C for long-term storage

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

• Buffer considerations:

  • The protein is typically supplied in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  • This buffer composition helps maintain protein stability

  • For specific applications, buffer exchange may be necessary but should be performed carefully to maintain protein integrity

How can researchers effectively measure COX6C enzymatic activity?

While COX6C itself is not an enzyme but a structural subunit, researchers can assess its contribution to cytochrome c oxidase function through several approaches:

• Cytochrome c oxidase activity assay:

  • Isolation of mitochondria from experimental cells (using differential centrifugation)

  • Preparation of a reaction mixture containing reduced cytochrome c

  • Measurement of the rate of cytochrome c oxidation at 550 nm

  • Calculation of activity based on the extinction coefficient of cytochrome c

• Comparative analysis with genetic manipulation:

  • Implement COX6C knockdown using siRNA or CRISPR/Cas9

  • Compare enzyme activity between normal and knockdown conditions

  • In a study with pancreatic cancer cells, COX6C knockdown led to measurable decreases in cytochrome c oxidase activity

• Polarographic methods:

  • Use of oxygen electrodes to measure oxygen consumption

  • Addition of specific substrates and inhibitors to isolate Complex IV activity

  • Normalization to protein content or cell number

• Data analysis and presentation:

  Condition	COX Activity (% of Control)	Statistical Significance
  Wild-type cells	100%	Reference
  Kras G12D cells	135%	P < 0.05
  Kras G12D + COX6C siRNA	85%	P < 0.01

  This table format can be used to present experimental results comparing cytochrome c oxidase activity under different conditions .

What techniques are suitable for detecting COX6C expression in tissue samples?

Several techniques are suitable for detecting COX6C expression in tissue samples, each with specific advantages:

• Immunohistochemistry (IHC):

  • Particularly valuable for tissue microarray analysis

  • Process: dewaxing, microwave antigen retrieval, endogenous peroxidase blocking, primary antibody incubation

  • Allows visualization of protein localization within cellular compartments

  • Enables scoring of expression levels (negative, weak, moderate, strong)

  • Can distinguish between cytoplasmic and other subcellular localizations

• Western blotting:

  • Quantitative assessment of protein expression

  • Requires protein extraction from tissue samples

  • Uses antibodies specific to COX6C

  • Typically normalized to loading controls such as GAPDH

  • Can detect differences in expression levels between sample types

• Quantitative PCR (qPCR):

  • Measures mRNA expression levels

  • Requires RNA extraction and reverse transcription

  • Can detect fold-changes in gene expression (e.g., 18.2-fold elevation observed in Kras G12D cells)

• In situ hybridization:

  • Detects mRNA within tissue sections

  • Preserves tissue architecture and cellular context

  • Complements protein detection methods

• Tissue microarray analysis:

  • Enables high-throughput analysis of multiple samples

  • Standardizes staining conditions across samples

  • Allows for comparative analysis between tumor and adjacent normal tissues

  • Useful for developing scoring systems for expression levels

How can COX6C function be inhibited in experimental settings?

Inhibiting COX6C function in experimental settings can be achieved through several approaches:

• RNA interference (RNAi):

  • siRNA targeting COX6C can effectively reduce its expression

  • Transfection of target cells with COX6C-specific siRNA

  • Verification of knockdown efficiency by Western blot or qPCR

  • Assessment of functional consequences (e.g., changes in enzyme activity, ATP production)

  • In pancreatic cancer research, this approach demonstrated that COX6C knockdown led to decreased cell viability and reduced cytochrome c oxidase activity

• CRISPR/Cas9 gene editing:

  • Generation of COX6C knockout or knockdown cell lines

  • Design of guide RNAs targeting COX6C gene

  • Selection and validation of edited clones

  • Functional characterization of resulting phenotypes

• Dominant negative mutants:

  • Expression of mutated forms of COX6C that interfere with normal function

  • Transfection of expression constructs encoding modified COX6C

  • Assessment of competitive inhibition of native protein function

• Small molecule inhibitors:

  • While specific inhibitors of COX6C are not widely available, inhibitors of cytochrome c oxidase complex can be used to study the consequences of disrupting the pathway

  • Examples include cyanide, azide, and carbon monoxide, though these affect the heme groups in catalytic subunits rather than specifically targeting COX6C

• Antibody-based inhibition:

  • For cell-free systems, specific antibodies against COX6C can be used to neutralize its function

  • This approach is limited to in vitro studies rather than cell-based assays

When implementing these inhibition strategies, researchers should include appropriate controls and carefully document the extent of inhibition achieved .

What are the best practices for comparative analysis of COX6C expression in different cell lines?

Conducting rigorous comparative analysis of COX6C expression across different cell lines requires adherence to several best practices:

• Standardized sample preparation:

  • Harvest cells at similar confluence levels (70-80% recommended)

  • Use consistent lysis buffers and extraction protocols

  • Process all samples simultaneously when possible

• Multiple detection methods:

  • Combine protein-level (Western blot) and mRNA-level (qPCR) analyses

  • Use consistent antibodies and primers across experiments

  • Include appropriate housekeeping genes or proteins as internal controls (e.g., GAPDH)

• Quantification approaches:

  • For Western blot: Use densitometry with normalization to loading controls

  • For qPCR: Apply the ΔΔCt method with validated reference genes

  • Present data as fold change relative to control cell lines

• Experimental design considerations:

  • Include biological replicates (minimum n=3)

  • Perform technical replicates for each biological sample

  • Use appropriate statistical tests to evaluate significance of differences

• Validation in multiple cell models:

  • Compare results across different cell lines from the same tissue type

  • Include normal and disease-state cell lines when relevant

• Functional correlation:

  • Link expression data with functional assays (enzyme activity, ATP production)

  • This approach was used successfully in pancreatic cancer research, where COX6C upregulation (4.6-fold at protein level) correlated with increased cytochrome c oxidase activity (35% increase)

• Data presentation:

  Cell Line	Relative mRNA Expression	Protein Expression (Fold Change)	COX Activity (% of Control)
  BxPC-3 (WT)	1.0	1.0	100%
  BxPC-3/Kras G12D	18.2	4.6	135%

  This table format effectively communicates the correlation between gene expression, protein levels, and functional consequences .

What metabolic pathways involve COX6C?

COX6C is involved in several critical metabolic pathways, functioning primarily as a component of the mitochondrial respiratory chain:

• Electron Transport Chain (ETC):

  • Primary pathway involving COX6C

  • Functions as part of Complex IV (cytochrome c oxidase)

  • Contributes to the terminal step of electron transfer from reduced cytochrome c to molecular oxygen

  • Works in conjunction with other ETC components including ATP5H, COX17, COX6A1, COX6A2, and COX7A2

• Oxidative Phosphorylation:

  • The electron transport facilitated by cytochrome c oxidase contributes to maintaining the proton gradient necessary for ATP synthesis

  • Indirectly supports ATP production via the established proton motive force

• Cardiac Muscle Contraction:

  • COX6C participates in pathways related to cardiac function

  • Works alongside proteins such as ACTC1A, MYL4, ATP2A2, and COX6A2 in this context

  • Supports energy production necessary for muscle contraction

• Metabolism:

  • Involved in broader metabolic networks

  • Interacts with components like COQ7, FABP9, and CEPT1

• Disease-related pathways:

  • Alzheimer's disease pathway: Interacts with components including ITPR3, NDUFA11, ATP5F1, and SDHD

  • Huntington's disease pathway: Functions alongside NDUFB11, CASP8, COX7A1, and ATP5O

Understanding these pathway involvements provides context for the diverse research applications of COX6C and explains its relevance to both normal physiological processes and disease states.

How is COX6C related to disease models?

COX6C has been implicated in several disease models, with research revealing important connections to pathological processes:

• Cancer models:

  • Upregulated in prostate cancer cells according to gene profiling studies

  • Significantly overexpressed in pancreatic cancer cells harboring the Kras G12D mutation (18.2-fold elevation at mRNA level, 4.6-fold at protein level)

  • Knockdown experiments demonstrate impacts on cancer cell viability, suggesting potential therapeutic implications

• Neurodegenerative disease models:

  • Involved in the Alzheimer's disease pathway

  • Associated with Huntington's disease pathway components

  • Mitochondrial dysfunction in these conditions may partially relate to alterations in cytochrome c oxidase function

• Cardiac disease models:

  • Participates in cardiac muscle contraction pathways

  • Alterations in COX6C and related proteins may contribute to energy metabolism deficiencies in cardiac pathologies

• Metabolic disorders:

  • As a component of mitochondrial respiratory function, COX6C may play roles in metabolic disease models characterized by bioenergetic dysfunction

  • Particularly relevant in conditions with impaired ATP production

• Expression in disease tissues:

  • Tissue microarray analysis has shown differential expression patterns between disease and normal tissues

  • In pancreatic cancer research, immunohistochemistry revealed distinct staining patterns between carcinoma and adjacent normal tissues

These relationships to disease models make COX6C a valuable target for both basic research into disease mechanisms and potential therapeutic development.

What is the relationship between COX6C and mitochondrial function?

COX6C plays an integral role in mitochondrial function through several mechanisms:

The relationship between COX6C and mitochondrial function highlights why this protein is important in contexts ranging from basic bioenergetics research to studies of diseases characterized by mitochondrial dysfunction.

How does COX6C expression correlate with ATP production?

Research evidence, particularly from cancer studies, demonstrates a significant correlation between COX6C expression and ATP production:

• Direct experimental evidence:

  • In pancreatic cancer research, BxPC-3/Kras G12D cells with upregulated COX6C showed enhanced ATP production compared to wild-type cells

  • When COX6C was knocked down using siRNA, a significant decrease in ATP production was observed

• Substrate utilization effects:

  • The correlation between COX6C expression and ATP production is substrate-dependent

  • Studies have examined ATP production in response to specific substrates:

    • Citrate (Cit)

    • α-ketoglutarate (AKG)

  • COX6C knockdown affected ATP production from these substrates differently

• Quantitative relationship:

  • ATP production can be measured using bioluminescent assays

  • Experimental protocols typically involve:

    • Isolation of mitochondria from approximately 10×10⁷ cells

    • Incubation with 10 mM substrate for 15 minutes at 37°C

    • Measurement of ATP levels using a bioluminescence assay

• Mechanistic basis:

  • The correlation between COX6C expression and ATP production is explained by its role in cytochrome c oxidase function

  • Enhanced cytochrome c oxidase activity contributes to more efficient electron transport

  • This in turn supports the proton gradient necessary for ATP synthesis via ATP synthase

• Experimental data representation:

  Condition	ATP Production (Relative Bioluminescence)	Statistical Significance
  Control cells + Citrate	100	Reference
  Kras G12D cells + Citrate	145	P < 0.05
  Kras G12D + COX6C siRNA + Citrate	85	P < 0.01

  This tabular format effectively illustrates the correlation between COX6C expression levels and ATP production capacity under different experimental conditions .

What research tools are available for studying COX6C interactions?

Researchers have access to several specialized tools and techniques for studying COX6C interactions:

• Recombinant proteins:

  • His-tagged recombinant COX6C proteins are commercially available

  • Full-length proteins from various species including Nycticebus coucang

  • These can be used for interaction studies, antibody production, and functional assays

• Antibody-based approaches:

  • Specific antibodies for detection of COX6C in Western blot, immunohistochemistry, and immunoprecipitation

  • Co-immunoprecipitation (Co-IP) to identify binding partners

  • Proximity ligation assays to detect in situ protein-protein interactions

• Genetic manipulation tools:

  • siRNA for transient knockdown of COX6C expression

  • CRISPR/Cas9 systems for gene editing to create knockout or tagged endogenous proteins

  • Expression vectors for overexpression studies or rescue experiments

• Protein interaction analysis:

  • Pull-down assays using tagged recombinant proteins

  • Yeast two-hybrid screening for identifying novel interaction partners

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

• Structural biology approaches:

  • X-ray crystallography of cytochrome c oxidase complex components

  • Cryo-electron microscopy for visualization of large protein complexes

  • Molecular dynamics simulations to predict interaction interfaces

• Cell and tissue-based resources:

  • Cell lines with differential COX6C expression (e.g., BxPC-3 vs. BxPC-3/Kras G12D)

  • Tissue microarrays for examining expression patterns in clinical samples

  • Engineered cell lines expressing tagged versions of COX6C

• Bioinformatic resources:

  • Protein interaction databases

  • Sequence analysis tools for comparing COX6C across species

  • Pathway analysis software to contextualize COX6C within broader networks

These tools collectively enable comprehensive investigation of COX6C interactions at molecular, cellular, and tissue levels.