Recombinant Pseudalopex griseus Cytochrome c oxidase subunit 2 (MT-CO2)

What is the functional significance of MT-CO2 in cellular respiration?

MT-CO2 functions as an essential component of the respiratory chain complex IV (cytochrome c oxidase), which catalyzes the final step in the electron transport chain—the reduction of oxygen to water. Specifically, MT-CO2 transfers electrons from cytochrome c via its binuclear copper A center to the bimetallic center of the catalytic subunit 1 . This electron transfer process is critical for:

• Maintaining the proton gradient across the inner mitochondrial membrane

• ATP synthesis through oxidative phosphorylation

• Oxygen consumption regulation in cellular respiration

• Metabolic adaptation during stress conditions

In Pseudalopex griseus, as in other mammals, MT-CO2's function is particularly crucial for high-energy demanding tissues such as cardiac and skeletal muscle, where efficient oxidative phosphorylation supports the energetic requirements of this active canid species.

What expression systems are most effective for producing recombinant Pseudalopex griseus MT-CO2?

Several expression systems can be utilized for producing recombinant MT-CO2, each with distinct advantages:

For Pseudalopex griseus MT-CO2, mammalian expression systems often produce the most functionally accurate recombinant protein due to proper post-translational modifications and folding. The methodological approach should involve:

• Gene synthesis with optimized codons for the selected expression system

• Incorporation of a purification tag (His6 or FLAG) at either terminus

• Verification of proper mitochondrial targeting sequences

• Expression in serum-free media for simplified downstream purification

• Affinity chromatography followed by size exclusion chromatography

How should researchers validate the structural integrity of recombinant MT-CO2?

Validating structural integrity requires a multi-analytical approach:

• SDS-PAGE and Western blotting: Compare migration pattern with native protein; use anti-MT-CO2 antibodies for verification

• Circular dichroism (CD) spectroscopy: Analyze secondary structure elements and compare with predicted models

  • Expected α-helical content: approximately 60-65%

  • β-sheet content: approximately 15-20%

• Mass spectrometry:

  • Confirm molecular weight (expected ~27,941 Da)

  • Peptide mapping to verify sequence coverage

  • Identification of post-translational modifications

• Differential scanning calorimetry (DSC): Assess thermal stability and domain folding

• Limited proteolysis: Examine accessibility of protease cleavage sites as indicator of proper folding

For recombinant Pseudalopex griseus MT-CO2, researchers should particularly focus on copper coordination sites, as these are critical for electron transfer function and may be compromised in improperly folded recombinant proteins.

What purification strategies are most effective for recombinant MT-CO2?

The optimal purification protocol involves multiple complementary techniques:

• Initial clarification:

  • Centrifugation (10,000 × g, 30 min, 4°C)

  • Filtration through 0.45 μm membrane

• Affinity chromatography:

  • For His-tagged constructs: Ni-NTA resin with imidazole gradient elution

  • For FLAG-tagged constructs: Anti-FLAG M2 affinity gel

• Ion exchange chromatography:

  • Anion exchange (Q Sepharose) at pH 8.0

  • Salt gradient: 50-500 mM NaCl

• Size exclusion chromatography:

  • Superdex 200 column in 20 mM Tris-HCl pH 7.5, 150 mM NaCl

• Quality control checkpoints:

  • Purity assessment: SDS-PAGE (≥95% purity)

  • Activity assessment: Cytochrome c oxidation assay

  • Endotoxin testing: LAL assay (<0.1 EU/mg protein)

This multi-step approach typically yields 2-5 mg of purified protein per liter of mammalian cell culture with preserved structural and functional properties.

What are the key buffer considerations for maintaining MT-CO2 stability?

Buffer composition critically affects MT-CO2 stability and function:

Long-term storage recommendations:

• Store at -80°C in small aliquots

• Include 10% glycerol to prevent freeze-thaw damage

• Limit freeze-thaw cycles to maximum 3

• For short-term storage (1-2 weeks), 4°C is preferable to freezing

How can researchers address post-translational modification challenges when working with recombinant Pseudalopex griseus MT-CO2?

Post-translational modifications (PTMs) represent a significant challenge in recombinant protein production. For MT-CO2, key considerations include:

• Identification of native PTMs in Pseudalopex griseus MT-CO2:

  • Perform mass spectrometry analysis of native protein from tissue samples

  • Compare with predicted PTMs from sequence analysis

  • Cross-reference with known PTMs in other canid species

• Selection of expression system based on PTM requirements:

  • For phosphorylation patterns: Mammalian cells (HEK293T)

  • For glycosylation: CHO cells or Pichia pastoris

  • For disulfide bond formation: Systems with oxidizing environments

• Engineered modifications:

  • Site-directed mutagenesis of key residues

  • Introduction of unnatural amino acids at PTM sites

  • Chemical modification post-purification

• Methodological approaches for PTM validation:

• Production of homogeneous PTM populations:

  • Co-expression with relevant modifying enzymes

  • In vitro enzymatic modification post-purification

  • Application of CRISPR-Cas9 to engineer host cell lines

For Pseudalopex griseus MT-CO2, special attention should be paid to the signal sequence processing by IMP1 as mentioned in the UniProt comments . This processing is critical for proper localization and function of the mature protein.

What methodological approaches can be used to study MT-CO2's role in metabolic adaptation under stress conditions?

Recent research indicates MT-CO2 is upregulated during glucose deprivation and facilitates glutaminolysis for tumor cell survival . To investigate this role in Pseudalopex griseus cells:

• In vitro cell culture models:

  • Primary cell isolation from Pseudalopex griseus tissues

  • Establishment of immortalized cell lines using SV40 large T antigen

  • Culture conditions mimicking metabolic stress (glucose deprivation, hypoxia)

• MT-CO2 expression manipulation:

  • CRISPR-Cas9 knockout or knockdown using siRNA/shRNA

  • Overexpression using lentiviral vectors

  • Inducible expression systems (Tet-On/Off)

• Metabolic flux analysis:

  • 13C-glutamine tracing to measure glutaminolysis rates

  • Seahorse XF analysis for oxygen consumption and extracellular acidification

  • Metabolomics profiling under various stress conditions

• Signaling pathway investigation:

  • Western blotting for Ras-MAPK pathway components

  • ChIP-seq for LSD1 binding at JUN promoter

  • RNA-seq for transcriptional changes

• Experimental design for integrated analysis:

Mechanistically, focus on the MT-CO2-mediated pathway where elevated MT-CO2 increases FAD levels, activating LSD1 to epigenetically upregulate JUN transcription, consequently promoting GLS1 and glutaminolysis .

How can researchers effectively investigate the interaction between MT-CO2 and other respiratory chain components?

Understanding MT-CO2's interactions within the respiratory chain requires sophisticated methodological approaches:

• Structural analysis techniques:

  • Cryo-electron microscopy of purified respiratory complexes

  • X-ray crystallography of MT-CO2 in complex with interacting partners

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Protein-protein interaction studies:

  • Co-immunoprecipitation with antibodies against MT-CO2 or interacting partners

  • Proximity labeling approaches (BioID, APEX) for in situ interaction mapping

  • FRET/BRET assays for real-time interaction monitoring in living cells

• Functional consequences of interactions:

  • Electron transfer kinetics using stopped-flow spectroscopy

  • Oxygen consumption measurements with specific inhibitors

  • Assembly analysis using blue native PAGE

• Comparative approach across species:

  • Sequence alignment of MT-CO2 from Pseudalopex griseus with other canids and mammalian species

  • Identification of conserved interaction motifs

  • Site-directed mutagenesis of putative interaction residues

• Super-complex assembly analysis:

  • Isolation of intact mitochondrial super-complexes using digitonin solubilization

  • Determination of complex stoichiometry using quantitative proteomics

  • Assessment of functional differences between free complex IV and super-complex-associated complex IV

A particularly valuable approach is the combination of genetic manipulation with structural and functional analyses. For example, researchers can express mutant forms of MT-CO2 in MT-CO2-depleted cells and measure both structural (via BN-PAGE) and functional (via respirometry) consequences.

What are the appropriate experimental designs for investigating MT-CO2's role in glutaminolysis regulation?

Building on the finding that MT-CO2 promotes glutaminolysis under glucose deprivation , a comprehensive experimental design should include:

• Baseline characterization:

  • Quantify native MT-CO2 expression levels across Pseudalopex griseus tissues

  • Measure basal glutaminase activity and glutamine dependence

  • Determine FAD/FADH₂ ratios in different metabolic states

• Genetic manipulation strategies:

  • CRISPR-Cas9 knockout of MT-CO2 and rescue experiments

  • Site-directed mutagenesis of FAD-interacting residues

  • LSD1 and JUN knockout/knockdown to validate the pathway

• Metabolic flux analysis:

  • 13C-glutamine tracing to track carbon flux through TCA cycle

  • Measurement of key metabolites (glutamate, α-ketoglutarate, succinate)

  • Glutaminase activity assays under various conditions

• Epigenetic regulation assessment:

  • ChIP-seq for LSD1 binding at JUN promoter and other targets

  • ATAC-seq to assess chromatin accessibility changes

  • H3K4 methylation status at target promoters

• Comprehensive experimental matrix:

• Validation in primary tissues:

  • Ex vivo tissue slice cultures from various Pseudalopex griseus organs

  • Comparison of tissue-specific metabolic adaptations

  • Correlation with physiological stress responses in the species

This comprehensive approach will help delineate the species-specific aspects of MT-CO2-mediated metabolic adaptation compared to the cancer cell models previously studied .

How does the amino acid sequence of Pseudalopex griseus MT-CO2 compare to other canid species, and what are the functional implications?

A comparative analysis of MT-CO2 sequences reveals important evolutionary and functional insights:

• Sequence comparison methodology:

  • Multiple sequence alignment (MSA) using MUSCLE or CLUSTAL algorithms

  • Phylogenetic tree construction using maximum likelihood methods

  • Calculation of selection pressures (dN/dS ratios) across the protein

• Key domains and motifs:

  • Copper-binding sites show highest conservation

  • Transmembrane domains exhibit species-specific variations

  • Interface residues interacting with other complex IV subunits

• Comparative analysis of canid species MT-CO2:

• Structure-function correlation:

  • Homology modeling based on mammalian cytochrome c oxidase crystal structures

  • Molecular dynamics simulations to predict stability differences

  • Docking studies with cytochrome c to assess species-specific interaction patterns

• Adaptive evolution analysis:

  • Identification of positively selected sites across canid lineages

  • Correlation with ecological and physiological adaptations

  • Metabolic rate comparisons between species

The sequence comparison indicates that while the catalytic core of MT-CO2 is highly conserved across canids, species-specific adaptations exist primarily in regions involved in membrane interactions and assembly of the respiratory complex. These differences likely reflect adaptations to varying metabolic demands across canid species with different hunting and activity patterns.