COX11 Antibody

What is COX11 and what is its primary function in mitochondria?

COX11 is a nuclear-encoded protein that functions primarily as a copper chaperone involved in the assembly of cytochrome c oxidase (COX, also known as Complex IV) of the mitochondrial respiratory chain. Rather than being a structural component of COX itself, COX11 plays a critical role in delivering copper to the complex, specifically to the Cu(B) center. The protein contains a transmembrane domain localized in the mitochondrial inner membrane .

Recent research has revealed that COX11 has dual functions: its primary role in COX assembly and an additional function in cellular redox homeostasis. Studies have shown that COX11 can provide antioxidative protection likely independent from its COX assembly function, suggesting it has evolved to help protect cells during heightened oxidative stress .

What disease associations have been identified for COX11 mutations?

Biallelic pathogenic variants in COX11 have been associated with infantile-onset mitochondrial encephalopathies. This gene-disease association was identified in two unrelated families using trio genome and exome sequencing. Patients with COX11 mutations presented with symptoms characteristic of primary mitochondrial diseases, which are genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects .

Functional studies demonstrated that fibroblasts from patients with COX11 mutations showed decreased ATP levels, particularly when glycolytic ATP generation was blocked. Importantly, this deficiency could be rescued by coenzyme Q10 (CoQ10) supplementation, suggesting a potential therapeutic approach for patients with COX11 variants .

How does COX11 structure relate to its function?

COX11 contains conserved cysteine residues that are critical for its function. For example, in Arabidopsis thaliana COX11 (AtCOX11), Cys219 and in Saccharomyces cerevisiae COX11 (ScCOX11), Cys208 are particularly important for its antioxidative protection function .

These cysteine residues can be oxidized by reactive oxygen species (ROS) and form inter- or intramolecular disulfide bridges. When these cysteines are mutated to alanines, the antioxidative function of COX11 is compromised. Interestingly, the variant where all three cysteines were mutated (ΔCys) showed instability and/or degradation, highlighting the importance of these residues for protein stability as well .

What are the recommended applications and dilutions for COX11 antibodies?

Based on validated data, COX11 antibodies can be used for multiple experimental applications with the following recommended dilutions:

It's important to note that optimal dilutions can be sample-dependent, and researchers should titrate the antibody in their specific testing systems to achieve optimal results .

What positive controls should be used when validating COX11 antibodies?

For validating COX11 antibodies, the following positive controls have been successfully used:

• For Western blot: Mouse liver tissue and mouse brain tissue have shown positive detection

• For immunoprecipitation: Mouse liver tissue has been validated

• For immunohistochemistry: Human pancreas cancer tissue has been verified as a suitable positive control

When selecting positive controls for your own experiments, consider using these validated tissues or cell lines where COX11 is known to be expressed.

How does COX11 contribute to cellular redox homeostasis beyond its role in COX assembly?

Research has revealed that COX11 plays a distinct role in protecting cells during oxidative stress, which appears to be evolutionarily conserved and independent of its function in COX complex assembly. Overexpression of putatively soluble COX11 variants substantially improved the resistance of yeast cells to the ROS inducer menadione, indicating an antioxidative protection function .

This protective function depends on specific conserved cysteine residues. For instance, Cys219 in AtCOX11 and Cys208 in ScCOX11 are particularly important for this antioxidative role. When these residues are mutated, the protective effect is diminished, suggesting these cysteines may act as ROS scavengers or participate in redox-sensing mechanisms .

Interestingly, growth retardation in yeast mutants under oxidative stress conditions does not seem to be linked to the status of the COX complex and cellular respiration, further supporting the idea that COX11's role in redox homeostasis is distinct from its COX assembly function .

What is the relationship between COX11 and coenzyme Q10 (CoQ10)?

A surprising finding in COX11 research is its connection to CoQ10. Studies have shown that ATP levels could be restored with CoQ10 supplementation in cells with COX11 deficiency. This is particularly intriguing since COX11 has no known role in CoQ10 biosynthesis .

The exact mechanism by which CoQ10 rescues ATP production in COX11-deficient cells remains unclear, but it represents an important area for further investigation and highlights a potential metabolic therapeutic approach for patients with COX11-related mitochondrial disease .

How do mutations in COX11 affect mitochondrial complex IV assembly and function?

Mutations in COX11 can impact mitochondrial complex IV (cytochrome c oxidase) assembly and function in several ways:

• Reduced complex IV assembly: Blue native Polyacrylamide Gel Electrophoresis (BN-PAGE) and immunoblot analysis of mitochondria from patient fibroblasts with COX11 mutations showed lower levels of assembled monomeric complex IV compared to controls. This was evident when using antibodies against multiple complex IV subunits (COX1, COX2, and COX4) .

• Decreased respiratory function: When glycolytic ATP generation was blocked with 2-deoxy-d-glucose, fibroblasts from patients with COX11 mutations showed markedly decreased ATP levels compared with control cells, indicating impaired respiratory function .

• Altered ROS levels: Interestingly, studies in both yeast and Arabidopsis models have shown that deletion or knockdown of COX11 resulted in reduced cellular ROS levels compared to wild-type strains. In yeast, the ΔSccox11 strain, which has a nonfunctional COX complex, showed a significant reduction in cellular ROS levels .

These findings suggest that COX11 mutations primarily affect complex IV assembly, leading to reduced respiratory function and altered cellular redox states.

What techniques are recommended for analyzing COX11-dependent complex IV assembly?

Several techniques have been validated for analyzing COX11-dependent complex IV assembly:

• Blue native Polyacrylamide Gel Electrophoresis (BN-PAGE): This technique can be used to analyze complex IV assembly in mitochondria isolated from cells. Typically, 30 μg of mitochondria are solubilized in either 1% Digitonin (to preserve supercomplex forms) or 1% Triton X-100 (to separate individual OXPHOS complexes into holoenzymes). Immunoblotting with antibodies against various complex IV subunits (COX1, COX2, COX4) can then reveal assembly defects .

• Quantitative immunoblotting: Densitometry of BN-PAGE data can be performed using software like ImageLab on the COX1 signal, taken as a ratio to Complex II succinate dehydrogenase complex subunit A. This provides quantitative assessment of complex IV assembly relative to other respiratory complexes .

• SDS-PAGE Western blot: Analysis of whole cell lysates by SDS-PAGE followed by Western blotting with antibodies against complex IV subunits (e.g., COXII) can complement BN-PAGE findings .

• OXPHOS enzyme activity assays: Measuring the activities of OXPHOS enzyme complexes (I, II, III, IV) and citrate synthase (CS) can provide functional information about complex IV and other respiratory complexes .

What methods can be used to assess the impact of COX11 on cellular ATP levels?

Multiple complementary methods have been successfully employed to assess the impact of COX11 on cellular ATP levels:

• Steady-state ATP level assays with different substrates: Cells can be incubated in media containing different substrates with or without inhibitors of specific metabolic pathways. For example, using 2-deoxy-d-glucose to block glycolytic ATP generation can help isolate the contribution of respiratory ATP production. Adding compounds like CoQ10 (50 μM) can test rescue effects .

• Luciferase-based ATP assays: These provide quantitative measurements of ATP content and can corroborate findings from other methods. They have been successfully used to demonstrate decreased respiration-derived ATP levels in COX11 patient cells and rescue by CoQ10 supplementation .

• FRET-based ATP sensors: These have been used in knockdown studies using CRISPR interference (CRISPRi) to show that COX11 is required to maintain mitochondrial-derived ATP levels .

When designing experiments to assess ATP levels, it's important to include appropriate controls and to distinguish between glycolytic and respiratory ATP production.

How can researchers assess the role of COX11 in oxidative stress responses?

Several methodological approaches have been validated for assessing COX11's role in oxidative stress responses:

• ROS detection with fluorescent dyes: DCFDA (2',7'-dichlorofluorescin diacetate) dye can be used to detect cellular ROS levels. Upon entering the cell and oxidation, this dye exhibits bright green fluorescence. This method has been used to show differences in ROS levels between wild-type and COX11-deficient cells, both under basal conditions and after treatment with oxidative stressors like paraquat .

• Oxidative stress tolerance assays: Growth of cells (particularly yeast models) on media containing ROS inducers like paraquat (PQ) or menadione can assess oxidative stress tolerance. For instance, overexpression of COX11 variants in wild-type yeast cells followed by growth tests in the presence or absence of oxidative stress (e.g., 120 μM menadione) can reveal protective effects .

• Gene expression analysis under oxidative stress: Quantitative PCR (qPCR) can be used to analyze COX11 transcript levels after treatment with oxidative reagents such as hydrogen peroxide (H2O2, 20 mM), tert-Butyl hydroperoxide (t-BOOH, 1 mM), or antimycin A (50 μM). This approach helps understand how COX11 expression responds to different types of oxidative stress .

• Cysteine mutant analysis: Creating COX11 variants where specific cysteine residues are mutated to alanines can help determine which cysteines are important for oxidative stress protection. Immunoblotting can then assess the stability and expression levels of these variants .

Why might COX11 antibodies show multiple bands in Western blots?

COX11 antibodies may show multiple bands in Western blots for several reasons:

• Multiple isoforms: Multiple transcript variants encoding different isoforms have been identified for the COX11 gene. These isoforms may have different molecular weights, resulting in multiple bands .

• Post-translational modifications: COX11 may undergo various post-translational modifications that alter its molecular weight or electrophoretic mobility.

• Dimerization or oligomerization: Research has observed higher molecular weight bands that could correspond to SDS- and β-mercaptoethanol-resistant COX11 dimers or oligomers. For instance, in addition to the main band of truncated ScCOX11 monomers (expected size 22.6 kDa), higher bands have been observed that could represent dimers or oligomers .

• Pseudogene expression: A related COX11 pseudogene has been identified on chromosome 6, which might produce protein products that cross-react with COX11 antibodies .

When interpreting Western blot results, it's important to compare the observed bands with the calculated molecular weight of COX11 (approximately 26kDa/31kDa) and to include appropriate positive and negative controls .

What factors might affect the detection of COX11 in immunohistochemistry applications?

Several factors can influence the detection of COX11 in immunohistochemistry:

• Antigen retrieval methods: For optimal results, it's recommended to use TE buffer pH 9.0 for antigen retrieval. Alternatively, citrate buffer pH 6.0 can also be used .

• Antibody dilution: The recommended dilution range for IHC is 1:20-1:200, but the optimal dilution may vary depending on the specific tissue and experimental conditions .

• Tissue type: Different tissues may express varying levels of COX11, affecting detection sensitivity. Human pancreas cancer tissue has been validated as a positive control for COX11 immunohistochemistry .

• Fixation method and duration: Overfixation can mask epitopes and reduce antibody binding, while inadequate fixation can compromise tissue morphology.

• Detection system sensitivity: The choice of detection system (e.g., DAB vs. fluorescent) can impact the sensitivity and specificity of COX11 detection.

To optimize COX11 detection in IHC applications, researchers should titrate the antibody concentration, compare different antigen retrieval methods, and include appropriate positive and negative controls.

What are the key unanswered questions regarding COX11's dual functions in mitochondria?

Several important questions remain unanswered regarding COX11's dual roles in COX assembly and redox homeostasis:

• Mechanism of CoQ10 rescue: The mechanism by which CoQ10 supplementation rescues ATP production in COX11-deficient cells is unclear, given that COX11 has no known role in CoQ10 biosynthesis. Understanding this mechanism could reveal new insights into mitochondrial energy metabolism and potential therapeutic approaches .

• Structural basis of redox function: While specific cysteine residues have been identified as important for COX11's antioxidative function, the structural basis of how these residues contribute to redox homeostasis remains to be fully elucidated .

• Compartmentalization of functions: How COX11 balances its dual roles in COX assembly and redox homeostasis, particularly regarding its localization within the mitochondria, requires further investigation.

• Clinical spectrum of COX11-related disorders: As COX11 mutations have only recently been linked to human disease, the full clinical spectrum of COX11-related disorders and potential genotype-phenotype correlations remain to be characterized .

• Therapeutic potential: Beyond CoQ10, exploration of other therapeutic approaches for COX11-related disorders could yield important clinical applications.

Addressing these questions will require integrated approaches combining structural biology, biochemistry, cell biology, and clinical research.