Recombinant Mouse Cytochrome c oxidase subunit 2 (Mtco2)

What is Recombinant Mouse Cytochrome c oxidase subunit 2 (Mtco2)?

Recombinant Mouse Cytochrome c oxidase subunit 2 (Mtco2), also known as COX2 or COII, is a mitochondrially-encoded protein that serves as a critical component of Complex IV (cytochrome c oxidase) in the electron transport chain. It is a 25,976 Da protein consisting of 227 amino acids with the sequence beginning with MAYPFQLGLQ . As a recombinant protein, it is artificially produced through genetic engineering techniques to enable research applications. This subunit plays a crucial role in cellular respiration by transferring electrons from cytochrome c via its binuclear copper A center to the bimetallic center of the catalytic subunit 1 .

How does Mtco2 function in the respiratory chain?

Mtco2 functions as an integral component of cytochrome c oxidase (Complex IV), which catalyzes the final step in the mitochondrial electron transport chain - the reduction of oxygen to water. Specifically:

• It receives electrons from reduced cytochrome c in the intermembrane space

• Transfers these electrons via its binuclear copper A (CuA) center

• Directs electrons to subunit 1's active site (binuclear center formed by heme A3 and copper B)

• This process ultimately reduces molecular oxygen using 4 electrons and 4 protons to produce 2 water molecules

This electron transfer is coupled to proton pumping across the inner mitochondrial membrane, contributing to the electrochemical gradient that drives ATP synthesis. Defects in MT-CO2 can lead to mitochondrial complex IV deficiency with diverse clinical manifestations ranging from isolated myopathy to severe multisystem disorders .

What experimental models are best suited for studying Mtco2 function?

When designing experiments to study Mtco2 function, researchers should consider multiple model systems depending on the specific research questions:

For investigating metabolic adaptations, glucose-deprived cancer cell lines have proven particularly valuable as they show robust upregulation of MT-CO2 expression, allowing researchers to study its role in metabolic reprogramming and stress response .

What are the most effective techniques for detecting and measuring Mtco2 expression?

Several complementary techniques can be employed to detect and quantify Mtco2 expression:

• Western Blotting: Mouse monoclonal antibodies specific to Mtco2 can be used for protein detection. The antibody [3G5F7G3] has been validated for this application .

• Immunocytochemistry/Immunofluorescence (ICC/IF): This technique allows visualization of Mtco2 localization within the mitochondria, particularly valuable for studying its membrane integration .

• Flow Cytometry: Validated antibodies can be used to quantify Mtco2 levels across different cell populations .

• qRT-PCR: For mRNA expression analysis, particularly useful when studying transcriptional regulation of MT-CO2 in response to stressors like glucose deprivation .

• Respirometry: Oxygen consumption measurements provide functional assessment of cytochrome c oxidase activity, which can be correlated with Mtco2 expression levels.

When designing such experiments, it's critical to include appropriate controls to account for mitochondrial mass variations between samples.

How can researchers effectively design experiments to study Mtco2 regulation under metabolic stress?

When designing experiments to investigate Mtco2 regulation under metabolic stress, researchers should follow these methodological steps:

• Define your variables clearly: For instance, if studying glucose deprivation, the independent variable would be glucose concentration and the dependent variable would be Mtco2 expression levels .

• Develop a specific, testable hypothesis: For example, "Glucose deprivation increases Mtco2 expression through activation of Ras signaling pathway" .

• Design treatments to manipulate the independent variable: Create a glucose concentration gradient (e.g., normal, low, and no glucose) in cell culture media .

• Group assignment: Use either between-subjects (different cell cultures for each condition) or within-subjects (same cell line measured at different time points) designs .

• Measurement plan: Determine how to measure Mtco2 expression (protein levels via Western blot, mRNA via qRT-PCR) and downstream effects (glutaminolysis markers, cell survival) .

Research has demonstrated that glucose deprivation leads to robust upregulation of MT-CO2 expression, making this an excellent model system for studying metabolic adaptation mechanisms .

How is Mtco2 involved in tumor cell adaptation to glucose deprivation?

Mtco2 plays a critical role in helping tumor cells adapt to glucose deprivation through several interconnected mechanisms:

• Upregulation in response to glucose deprivation: Studies have shown that glucose deprivation, a common condition in the tumor microenvironment, triggers robust upregulation of MT-CO2 expression .

• Promotion of alternative energy pathways: Elevated MT-CO2 facilitates glutaminolysis, allowing tumor cells to utilize glutamine as an alternative energy source when glucose is limited .

• Molecular mechanism: The process involves:

  • Activation of Ras signaling to enhance MT-CO2 transcription

  • Inhibition of IGF2BP3 (an RNA-binding protein) to stabilize MT-CO2 mRNA

  • Increased FAD levels that activate lysine-specific demethylase 1 (LSD1)

  • Epigenetic upregulation of JUN transcription

  • Promotion of glutaminase-1 (GLS1) expression and glutaminolysis

This adaptation mechanism is particularly important in solid tumors where glucose concentrations are typically 3-10 fold lower than in normal tissues .

What is the relationship between Mtco2, Ras signaling, and cancer progression?

The relationship between Mtco2, Ras signaling, and cancer progression represents a significant research area with therapeutic implications:

• Bidirectional relationship: Ras signaling enhances MT-CO2 transcription, while MT-CO2 is essential for oncogenic Ras-induced glutaminolysis and tumor growth .

• Clinical significance: Elevated expression of MT-CO2 is associated with poor prognosis in lung cancer patients, indicating its potential value as a prognostic marker .

• Therapeutic target: MT-CO2 has been highlighted as a putative therapeutic target specifically for Ras-driven cancers, which are notoriously difficult to treat .

• Metabolic rewiring: This relationship demonstrates how mitochondrial-encoded proteins can participate in metabolic reprogramming to support tumor growth under nutrient-restricted conditions .

Researchers investigating this pathway should consider experimental designs that manipulate both Ras signaling and Mtco2 expression to fully characterize their interdependence in cancer models.

How do post-translational modifications affect Mtco2 function and stability?

Post-translational modifications (PTMs) of Mtco2 represent an understudied area with significant implications for understanding respiratory chain function. When investigating PTMs of Mtco2, researchers should consider:

• Identification methods: Mass spectrometry-based proteomic approaches are the gold standard for identifying PTMs. Special sample preparation techniques are required for membrane proteins like Mtco2.

• Common modifications to investigate:

  • Phosphorylation sites that may regulate protein-protein interactions

  • Acetylation that might respond to metabolic states

  • Oxidative modifications that could indicate damage or signaling

• Functional assessment: After identifying modifications, site-directed mutagenesis can be used to create phospho-mimetic or phospho-resistant mutations to assess functional consequences on enzyme activity.

• Regulatory enzymes: Identifying the kinases, acetyltransferases, or other enzymes that modify Mtco2 will provide insights into regulatory networks.

While current literature on Mtco2-specific PTMs is limited, this represents a promising area for future research, particularly in understanding how these modifications might mediate rapid adaptations to metabolic stress.

What methodologies are most effective for studying the role of Mtco2 in the context of mitochondrial diseases?

When investigating Mtco2's role in mitochondrial diseases, researchers should employ these methodological approaches:

• Patient-derived samples: Analysis of MT-CO2 mutations in patients with mitochondrial complex IV deficiency provides direct clinical relevance. Researchers should consider:

  • Sequencing of mitochondrial DNA

  • Biochemical assessment of complex IV activity

  • Structural analysis of how mutations affect protein function

• Disease models: Several complementary models can be employed:

  • Cybrid cell lines (patient mitochondria in control nuclear background)

  • CRISPR-engineered cell lines with specific MT-CO2 mutations

  • Mouse models with mutations corresponding to human disease variants

• Functional assessments: Beyond measuring complex IV activity, researchers should evaluate:

  • Mitochondrial membrane potential

  • ATP production capacity

  • Reactive oxygen species generation

  • Cell viability under various stressors

Defects in MT-CO2 can cause mitochondrial complex IV deficiency with heterogeneous clinical manifestations ranging from isolated myopathy to severe multisystem disease affecting several tissues and organs . Research using these methodologies can help clarify genotype-phenotype correlations.