Recombinant Trachypithecus cristatus Cytochrome c oxidase subunit 6C (COX6C)

What is Trachypithecus cristatus COX6C and what is its functional significance?

Trachypithecus cristatus (silvered leaf-monkey) COX6C is a nuclear-encoded structural subunit of cytochrome c oxidase (Complex IV), the terminal enzyme in the mitochondrial electron transport chain. This subunit plays a crucial role in the regulation of oxidative phosphorylation (OXPHOS). The protein consists of approximately 75 amino acids and is involved in electron transfer from reduced cytochrome c to molecular oxygen, ultimately contributing to ATP production .

Unlike several other COX subunits, COX6C has no paralogs in any vertebrate lineage, including mammals, suggesting its evolutionarily conserved and non-redundant function . The mature protein shows significant homology between species, with approximately 69-73% sequence similarity when comparing rat, bovine, and human variants, indicating its functional importance across mammalian species .

When studying T. cristatus COX6C, researchers should note that it functions within the respiratory chain that contains three multisubunit complexes: succinate dehydrogenase (Complex II), ubiquinol-cytochrome c oxidoreductase (Complex III), and cytochrome c oxidase (Complex IV) . These complexes cooperatively transfer electrons to create an electrochemical gradient that drives ATP synthesis.

How does one design expression vectors for recombinant Trachypithecus cristatus COX6C production?

Designing expression vectors for recombinant T. cristatus COX6C requires careful consideration of multiple factors:

• Gene sequence optimization: The COX6C gene sequence should be codon-optimized for the selected expression system. This typically involves:

  • Adapting codon usage bias to match the expression host

  • Removing cryptic splice sites and unwanted regulatory elements

  • Optimizing GC content and removing repetitive sequences

• Vector selection: Consider vectors with:

  • Strong, inducible promoters like T7 for bacterial systems or CMV for mammalian cells

  • Appropriate selection markers (antibiotic resistance genes)

  • Fusion tags for purification (His, GST, or Fc tags)

  • Signal peptides if secretion is desired

• Affinity tag placement: Based on structural considerations and the nature of COX6C:

  • N-terminal tags are often preferred due to the membrane-associating properties of COX6C

  • Inclusion of a protease cleavage site between the tag and COX6C

  • Consider TEV or PreScission protease sites for tag removal

When designing your constructs, it's crucial to verify the full 933-bp linear DNA sequence of COX6C to ensure all functional domains are preserved . Given that COX6C is typically located at chromosome 8q22.2 in humans (with potential variations in T. cristatus), proper gene annotation verification is essential before proceeding with cloning .

What expression systems are most effective for producing functional recombinant Trachypithecus cristatus COX6C?

Several expression systems have been employed for recombinant COX6C production, each with distinct advantages:

For T. cristatus COX6C specifically, wheat germ cell-free systems have shown success with human COX6C and would likely be appropriate for the closely related T. cristatus variant . This system preserves the protein in the 1 to 75 amino acid range with high fidelity to the native structure.

When selecting an expression system, consider the downstream applications. For interaction studies with other mitochondrial proteins, mammalian expression may be preferable to maintain physiologically relevant modifications. For structural studies requiring large protein quantities, E. coli or insect cell systems might be more appropriate.

What purification strategies yield high-purity recombinant Trachypithecus cristatus COX6C?

Purification of recombinant T. cristatus COX6C requires a multi-step approach to achieve high purity while maintaining protein functionality:

• Initial capture: Affinity chromatography based on the fusion tag:

  • His-tagged COX6C: Ni-NTA or IMAC (Immobilized Metal Affinity Chromatography)

  • GST-tagged COX6C: Glutathione Sepharose

  • Fc-tagged COX6C: Protein A/G columns

• Intermediate purification:

  • Ion exchange chromatography (IEX): Based on COX6C's theoretical isoelectric point

  • Size exclusion chromatography (SEC): To separate monomeric COX6C from aggregates

  • Tag cleavage: Using specific proteases like TEV or PreScission

• Polishing step:

  • Reverse-phase HPLC for highest purity

  • Second affinity step to remove cleaved tags

A typical purification protocol might include:

• Lysis in a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% detergent (if membrane-associated), and protease inhibitors

• Affinity purification using the appropriate resin

• Buffer exchange to remove imidazole or glutathione

• Overnight tag cleavage at 4°C

• Second affinity step to remove cleaved tag

• Size exclusion chromatography for final polishing

Throughout purification, it's crucial to monitor COX6C by SDS-PAGE and Western blotting using antibodies against COX6C or the fusion tag . Activity assays measuring cytochrome-c oxidase activity should be performed to confirm functionality .

How can researchers verify the structural integrity and functionality of purified recombinant Trachypithecus cristatus COX6C?

Verification of recombinant T. cristatus COX6C requires comprehensive characterization:

• Structural integrity assessment:

  • Mass spectrometry (MS): To confirm the exact molecular weight

  • Circular dichroism (CD): To evaluate secondary structure content

  • Fluorescence spectroscopy: To assess tertiary structure

  • Limited proteolysis: To determine domain stability

  • Dynamic light scattering (DLS): To check homogeneity and aggregation state

• Functional verification:

  • Cytochrome-c oxidase activity assay: Measuring electron transfer rates

  • Oxygen consumption assays: Using Clark-type electrodes

  • Protein-protein interaction assays: With known partners like MT-CO1, MT-CO2, or COX5A

  • Reconstitution assays: Incorporation into artificial membranes to measure activity

• Immunological confirmation:

  • Western blotting with anti-COX6C antibodies

  • Immunoprecipitation with interaction partners

  • ELISA for quantitative assessment

A functional COX6C should demonstrate:

• Proper integration into the cytochrome c oxidase complex

• Contribution to electron transfer from cytochrome c to oxygen

• Expected protein-protein interactions with other subunits

• Correct subcellular localization to mitochondria when expressed in cells

Given COX6C's role in respiratory function, oxidative phosphorylation activity measurements in reconstituted systems provide the most direct assessment of functionality .

What methodologies are optimal for studying the interactome of Trachypithecus cristatus COX6C?

Studying the COX6C interactome requires sophisticated approaches to capture both stable and transient interactions:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged T. cristatus COX6C in relevant cell lines

  • Perform gentle cell lysis preserving native interactions

  • Capture COX6C and associated proteins via affinity purification

  • Identify interacting partners through LC-MS/MS

  • Compare to control pulldowns to eliminate false positives

• Proximity-based labeling techniques:

  • BioID: Fusion of COX6C with a promiscuous biotin ligase

  • APEX2: Fusion with an engineered ascorbate peroxidase

  • TurboID: Using an improved biotin ligase for faster labeling

  • These approaches allow identification of proteins in close proximity to COX6C in living cells

• Crosslinking mass spectrometry (XL-MS):

  • Use homo- or hetero-bifunctional crosslinkers

  • Identify crosslinked peptides by MS to map interaction interfaces

  • Particularly valuable for membrane protein complexes like cytochrome c oxidase

Known interacting partners of COX6C include MT-CO1, MT-CO2, COX5A, and other components of the cytochrome c oxidase complex . Additionally, interactions with regulatory proteins such as SURF1 and LRRK2 have been reported . These established interactions serve as positive controls for new interactome studies with T. cristatus COX6C.

The interactome data can be analyzed using:

• Protein interaction network visualization tools

• Functional enrichment analysis

• Evolutionary conservation mapping across primate species

• Integration with pathway databases focused on mitochondrial function

How can researchers investigate the tissue-specific expression patterns of Trachypithecus cristatus COX6C?

Investigating tissue-specific expression patterns of T. cristatus COX6C requires a multi-faceted approach:

• Transcriptional profiling:

  • RNA-Seq analysis across different T. cristatus tissues

  • qRT-PCR validation of expression levels using tissue-specific samples

  • Single-cell RNA-Seq to identify cell-type-specific expression

  • Comparison with expression patterns in other primates for evolutionary insights

• Protein-level detection:

  • Immunohistochemistry (IHC) on tissue sections using COX6C-specific antibodies

  • Western blotting of tissue lysates with quantitative analysis

  • Targeted proteomics (SRM/MRM) for absolute quantification

  • Analysis of post-translational modifications across tissues

• Regulatory mechanism investigation:

  • Chromatin immunoprecipitation (ChIP) to identify transcription factors

  • Promoter analysis for tissue-specific regulatory elements

  • ATAC-Seq to assess chromatin accessibility in different tissues

  • Analysis of potential microRNA regulation, considering known regulators like miR-4276

Based on studies in other species, COX6C expression may vary significantly across tissues, with particularly high expression in metabolically active tissues like heart, brain, and skeletal muscle . Mitochondrial biogenesis in response to environmental factors such as temperature can also influence COX6C expression levels .

A comparative analysis approach should include:

• Correlation of expression with tissue-specific metabolic demands

• Examination of co-regulated genes in the OXPHOS pathway

• Analysis of expression changes during development and aging

• Comparison with human expression patterns to identify primate-specific regulation

Post-translational modifications (PTMs) of T. cristatus COX6C can be comprehensively studied using:

• Mass spectrometry-based approaches:

  • Bottom-up proteomics: Enzymatic digestion followed by LC-MS/MS

  • Top-down proteomics: Analysis of intact protein to preserve PTM combinations

  • Middle-down approach: Limited proteolysis for analysis of larger fragments

  • Targeted MS methods: SRM/MRM for quantification of specific modifications

• Enrichment strategies for specific PTMs:

  • Phosphorylation: TiO2, IMAC, or phospho-specific antibodies

  • Acetylation: Anti-acetyl lysine antibodies

  • Ubiquitination: Tandem ubiquitin binding entities (TUBEs)

  • Oxidative modifications: Derivatization approaches (e.g., dimedone for sulfenic acids)

• Functional impact assessment:

  • Site-directed mutagenesis of modified residues

  • In vitro enzymatic assays with modified and unmodified protein

  • Structural analysis using NMR or X-ray crystallography

  • Molecular dynamics simulations to predict PTM effects on protein dynamics

While specific PTMs of T. cristatus COX6C are not well-characterized, studies in human COX6C suggest potential regulatory modifications. The human protein shows evidence of regulation through splicing efficiency by DAZAP1, which may involve downstream PTM regulation .

A comprehensive PTM analysis workflow should include:

• Careful sample preparation to preserve labile modifications

• Parallel analysis of samples from different physiological conditions

• Validation of identified PTMs using orthogonal methods

• Functional studies to determine the impact on COX6C activity

• Evolutionary comparison of modification sites across primate species

How can researchers study the evolutionary conservation of COX6C across primates including Trachypithecus cristatus?

Studying evolutionary conservation of COX6C across primates requires:

• Comparative genomic analysis:

  • Multiple sequence alignment of COX6C coding sequences

  • Phylogenetic tree construction using maximum likelihood or Bayesian methods

  • Calculation of selection pressure using dN/dS ratios

  • Identification of conserved domains and species-specific variations

  • Synteny analysis of the genomic region containing COX6C

• Structural and functional comparison:

  • Homology modeling of T. cristatus COX6C based on available structures

  • Identification of functionally important residues across species

  • Analysis of protein-protein interaction interfaces

  • Prediction of functional consequences of species-specific variations

• Experimental validation:

  • Recombinant expression of COX6C from multiple primate species

  • Comparative functional assays for cytochrome c oxidase activity

  • Cross-species complementation experiments

  • Chimeric protein studies to identify functionally divergent regions

COX6C is notable for having no paralogs in any vertebrate lineage, suggesting strong evolutionary constraints . The mature protein shows significant sequence conservation with approximately 69-73% homology between rats, bovines, and humans .

Research should focus on:

• Correlation between evolutionary changes and primate-specific metabolic adaptations

• Comparison of regulatory mechanisms across primate species

• Analysis of COX6C in relation to chromosomal rearrangements in primate evolution

• Integration of findings with broader studies of mitochondrial evolution in primates

What methodologies are most effective for studying the role of Trachypithecus cristatus COX6C in response to environmental stressors?

Investigating the role of T. cristatus COX6C in response to environmental stressors requires:

• Experimental stress models:

  • Hypoxia chambers with controlled oxygen levels

  • Temperature variation experiments (both hypo- and hyperthermia)

  • Oxidative stress induction using H₂O₂ or paraquat

  • Nutrient deprivation (glucose or amino acid restriction)

  • Viral infection models, particularly influenza virus exposure

• Multi-omics approaches:

  • Transcriptomics: RNA-Seq to monitor COX6C expression changes

  • Proteomics: Quantitative analysis of COX6C and interacting proteins

  • Metabolomics: Assessment of changes in OXPHOS metabolites

  • Integrated pathway analysis combining multiple data types

• Dynamic measurements:

  • Real-time quantification of COX6C expression using reporter constructs

  • Live-cell imaging of mitochondrial function

  • Temporal analysis of stress response pathways

  • Recovery kinetics after stress removal

Studies have shown that COX6C expression can be modulated by various stressors. For example, mitochondrial biogenesis stimulated by low temperature is reflected in increased expression and activity of COX6C . During viral infections, particularly influenza, host cells initially boost COX6C mRNA expression by silencing miR-4276 as a defense mechanism .

A comprehensive experimental design should include:

• Time-course experiments to capture both immediate and adaptive responses

• Dose-response studies to determine threshold effects

• Combined stressors to model complex environmental conditions

• Parallel analysis in multiple cell types or tissues

• Comparison with responses in human or other primate cells

What are the key methodological challenges in structural studies of Trachypithecus cristatus COX6C?

Structural characterization of T. cristatus COX6C presents several technical challenges:

• Protein production challenges:

  • Maintaining structural integrity during recombinant expression

  • Ensuring proper membrane association and folding

  • Obtaining sufficient quantities for structural studies

  • Preventing aggregation of this hydrophobic protein

• Crystallization barriers:

  • Difficulty in crystallizing membrane-associated proteins

  • Need for appropriate detergents or lipid environments

  • Challenge of obtaining well-diffracting crystals

  • Requirement for stabilizing protein-protein interactions

• Alternative structural approaches:

  • Cryo-EM for structure determination within the intact cytochrome c oxidase complex

  • Solution NMR for dynamic studies of isolated domains

  • Small-angle X-ray scattering (SAXS) for low-resolution envelope determination

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

• Computational methods:

  • Molecular dynamics simulations in membrane environments

  • Ab initio structure prediction using deep learning approaches

  • Integrative modeling combining multiple experimental data sources

  • Evolutionary coupling analysis to predict structural contacts

The most effective approach is likely to be integrative, combining:

• High-resolution structural techniques for isolated domains

• Cryo-EM for the intact cytochrome c oxidase complex

• Crosslinking mass spectrometry to map interaction interfaces

• Computational modeling to fill gaps in experimental data

Researchers should consider that COX6C functions as part of the larger cytochrome c oxidase complex, which includes multiple subunits that cooperatively catalyze the reduction of oxygen to water . Structural studies should aim to understand both the isolated protein and its role within this larger complex.

How can researchers optimize assays to measure the enzymatic activity of recombinant Trachypithecus cristatus COX6C?

Optimizing enzymatic activity assays for recombinant T. cristatus COX6C requires careful consideration of multiple factors:

• Direct activity measurement approaches:

  • Oxygen consumption assays using Clark-type electrodes

  • Spectrophotometric assays monitoring cytochrome c oxidation

  • Polarographic methods to measure electron transfer rates

  • Artificial electron acceptor/donor systems for isolated protein studies

• Reconstitution strategies:

  • Incorporation into liposomes or nanodiscs

  • Co-expression with other cytochrome c oxidase subunits

  • Stepwise assembly of the complex in vitro

  • Integration into mitochondrial membrane fractions

• Assay optimization parameters:

  • Buffer composition (pH, ionic strength, divalent cations)

  • Detergent type and concentration

  • Lipid composition of reconstitution systems

  • Temperature and reaction time course

  • Substrate concentrations (reduced cytochrome c)

• Controls and validation:

  • Specific inhibitors (e.g., cyanide, azide) for validation

  • Comparison with native enzyme activity

  • Mutational analysis of key residues

  • Competition assays with purified COX6C

A comprehensive enzymatic characterization should include:

• Determination of kinetic parameters (Km, Vmax, kcat)

• Effects of physiological regulators and inhibitors

• pH and temperature profiles

• Stability studies under various conditions

• Comparison with human COX6C activity

Given the role of COX6C in electron transfer and the reduction of oxygen to water , researchers should ensure that activity assays effectively capture this specific function within the context of the complete cytochrome c oxidase complex.

What experimental approaches can resolve contradictions in published data about COX6C function?

Resolving contradictions in COX6C functional data requires systematic approaches:

• Standardized methodology development:

  • Establish consensus protocols for COX6C expression and purification

  • Define standard activity assay conditions

  • Create reference datasets for benchmarking

  • Develop validated antibodies and detection methods

• Multi-laboratory validation studies:

  • Collaborative projects testing the same hypotheses

  • Round-robin testing of identical protein preparations

  • Meta-analysis of published data with rigorous quality assessment

  • Pre-registered replication studies of key findings

• Addressing specific contradictions:

  • Species-specific differences: Systematic comparison across species including T. cristatus

  • Isoform-specific effects: Careful characterization of all potential isoforms

  • Context-dependent functions: Testing in multiple cellular/tissue environments

  • Regulatory discrepancies: Comprehensive analysis of regulatory pathways

• Advanced technologies to provide new perspectives:

  • Single-molecule techniques to address population heterogeneity

  • In situ structural studies using cryo-electron tomography

  • Systems biology approaches integrating multiple data types

  • CRISPR screening for genetic interactions

When examining contradictory findings, researchers should consider:

• The dual role of COX6C in electron transport and potential regulatory functions

• The influence of experimental conditions on membrane protein behavior

• Tissue-specific variations in function and regulation

• Potential differences between recombinant and native proteins

For example, contradictory findings regarding COX6C in disease models (such as its upregulation in diabetic nephropathy but downregulation in end-stage renal disease) can be addressed by careful time-course studies and consideration of disease progression stages.

How can Trachypithecus cristatus COX6C be used as a model for understanding primate-specific aspects of mitochondrial function?

T. cristatus COX6C offers unique opportunities for understanding primate-specific mitochondrial biology:

• Comparative studies across primates:

  • Functional comparison with human and other primate COX6C

  • Analysis of primate-specific adaptations in mitochondrial function

  • Investigation of lineage-specific selection pressures

  • Integration with broader studies of primate evolution

• Experimental model development:

  • T. cristatus cell lines for mitochondrial studies

  • CRISPR-engineered human cells expressing T. cristatus COX6C

  • Hybrid mitochondrial complexes with components from different species

  • Computational models of species-specific mitochondrial function

• Evolutionary medicine applications:

  • Insights into primate-specific mitochondrial disorders

  • Understanding adaptive changes in OXPHOS efficiency

  • Investigation of metabolic adaptations to different environments

  • Analysis of mitochondrial contributions to primate longevity

T. cristatus represents an interesting evolutionary position, with its COX6C showing significant homology to human COX6C but with species-specific variations. The silvered leaf monkey has also been the subject of detailed chromosomal homology studies with humans, which can provide context for understanding the genomic environment of COX6C .

Future research directions could include:

• The role of COX6C in primate-specific metabolic adaptations

• Comparative analysis of regulatory mechanisms across primates

• Investigation of mitochondrial-nuclear co-evolution in primates

• Development of primate-specific mitochondrial disease models

What are the emerging technologies that will advance our understanding of Trachypithecus cristatus COX6C?

Several cutting-edge technologies promise to revolutionize COX6C research:

• Advanced imaging techniques:

  • Super-resolution microscopy for subunit localization within complexes

  • Single-particle cryo-EM for high-resolution structural studies

  • Correlative light and electron microscopy (CLEM) for structure-function studies

  • Live-cell imaging with genetically encoded sensors

• Next-generation genomic and transcriptomic approaches:

  • Long-read sequencing for complete locus characterization

  • Single-cell multi-omics for cell-type-specific analysis

  • Spatial transcriptomics for tissue-specific expression patterns

  • CRISPR screens for functional genomics

• Protein engineering and synthetic biology:

  • Directed evolution of COX6C variants with enhanced properties

  • De novo design of artificial COX6C with novel functions

  • Optogenetic control of COX6C activity

  • Engineered allosteric regulators of COX6C function

• Computational and AI approaches:

  • AlphaFold2 and RoseTTAFold for structure prediction

  • Machine learning for predicting functional effects of mutations

  • Systems biology models of mitochondrial function

  • Network analysis of mitochondrial protein interactions

These technologies will enable:

• Higher resolution understanding of COX6C's role within Complex IV

• More precise manipulation of COX6C function in experimental systems

• Better integration of structural, functional, and evolutionary data

• Development of new therapeutic approaches targeting COX6C-related pathways

As research progresses, integration of data across multiple technologies will be crucial for a comprehensive understanding of T. cristatus COX6C biology and its relevance to human mitochondrial function and disease.