Recombinant Tarsius syrichta Cytochrome c oxidase subunit 6C (COX6C)

What is Cytochrome c oxidase subunit 6C and what is its role in cellular respiration?

Cytochrome c oxidase subunit 6C (COX6C) is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport. As a component of the cytochrome c oxidase (Complex IV), it plays a crucial role in the respiratory chain that catalyzes the reduction of oxygen to water. The respiratory chain contains three multisubunit complexes that cooperate to transfer electrons derived from NADH and succinate to molecular oxygen, creating an electrochemical gradient over the inner membrane that drives transmembrane transport and ATP synthesis .

In this process, electrons originating from reduced cytochrome c in the intermembrane space are transferred via the dinuclear copper A center of subunit 2 and heme A of subunit 1 to the active site in subunit 1, which is formed by heme A3 and copper B. This binuclear center reduces molecular oxygen to water molecules using electrons from cytochrome c and protons from the mitochondrial matrix .

What is the amino acid sequence of Tarsius syrichta COX6C?

The amino acid sequence of Tarsius syrichta Cytochrome c oxidase subunit 6C is:

SSGALTKPQMRGLLAKRLRFHIVGAFAVSLGVAAFYKFAVAEPRKKAYADFYRNYDSMKDFEEMRKAGIFQSAK

This sequence represents the full protein, with the expression region typically spanning amino acids 2-75 .

How is Tarsius syrichta COX6C different from human COX6C?

While the search results don't provide a direct sequence comparison between Tarsius syrichta and human COX6C, we can infer from the available data that there are sufficient structural and functional similarities to allow for comparative studies. Human COX6C has a calculated molecular weight of approximately 9 kDa and consists of 75 amino acids , which is comparable to the Tarsius syrichta COX6C specifications.

What are the optimal conditions for expression and purification of recombinant Tarsius syrichta COX6C?

Based on the available information, recombinant Tarsius syrichta COX6C can be successfully expressed in yeast expression systems with a purity of >85% as confirmed by SDS-PAGE . The recombinant protein is typically produced as a partial construct, with the expression region spanning amino acids 2-75 .

For optimal expression and purification:

• Expression System: Yeast expression systems appear to be effective for producing functional recombinant Tarsius syrichta COX6C .

• Purification Method: While specific details are not provided in the search results, standard affinity chromatography methods are likely applicable, particularly if the recombinant protein includes an affinity tag. The tag type is typically determined during the production process to optimize protein stability and function .

• Storage Buffer: The recombinant protein is typically stored in a Tris-based buffer with 50% glycerol, which has been optimized for protein stability .

How can researchers address challenges in studying mitochondrial proteins from endangered species like Tarsius syrichta?

Studying mitochondrial proteins from endangered species like the Philippine tarsier (Tarsius syrichta) presents unique challenges due to limited sample availability and conservation concerns. Researchers can address these challenges through several approaches:

• Non-invasive Sampling: Given that Tarsius syrichta is threatened and endangered , researchers should prioritize non-invasive sampling methods when obtaining genetic material. Conservation genetics studies of Tarsius syrichta have successfully used QIAGEN DNeasy extraction kits for DNA extraction from minimal tissue samples .

• Recombinant Protein Technology: Using recombinant protein technology, as exemplified by the production of recombinant Tarsius syrichta COX6C in yeast systems , allows researchers to study the protein's properties without requiring additional samples from wild populations.

• Comparative Genomics: Leveraging the genetic diversity data available from conservation genetics studies can provide context for understanding the variation in mitochondrial proteins across different island populations of Tarsius syrichta.

• Collaborative Conservation Efforts: Research on Tarsius syrichta should be conducted in the context of conservation efforts, as captive breeding attempts have been largely unsuccessful, and these animals are significantly affected by deforestation and illegal pet trade .

What methodological approaches can be used to study the interaction between COX6C and other subunits of the cytochrome c oxidase complex?

To study the interactions between COX6C and other subunits of the cytochrome c oxidase complex, researchers can employ several methodological approaches:

• Co-immunoprecipitation (Co-IP): Using available antibodies against COX6C , researchers can perform Co-IP experiments to identify protein-protein interactions within the complex. The polyclonal antibody (11429-2-AP) has been validated for Western Blot applications and could be used to pull down COX6C along with its interacting partners.

• Crosslinking Mass Spectrometry: This technique can capture transient interactions and provide detailed information about the spatial arrangement of subunits within the complex.

• Blue Native PAGE: This technique allows for the separation of intact protein complexes under native conditions, enabling the study of COX6C within the context of the entire cytochrome c oxidase complex.

• Cryo-electron Microscopy: This structural biology technique can provide high-resolution insights into the architecture of the cytochrome c oxidase complex, including the positioning of COX6C relative to other subunits.

What are the recommended protocols for using antibodies against COX6C in Western Blot and Immunohistochemistry applications?

For Western Blot (WB) applications:

• Dilution Ratio: Use antibodies against COX6C at a dilution ratio of 1:500-1:1000 .

• Sample Preparation: Human heart tissue has been validated as a positive control for WB applications .

• Detection Method: Follow standard secondary antibody and detection protocols as provided in the WB protocol for COX6C antibody 11429-2-AP .

For Immunohistochemistry (IHC) applications:

• Dilution Ratio: Use antibodies at a dilution ratio of 1:20-1:200 .

• Antigen Retrieval: Suggested antigen retrieval should be performed with TE buffer pH 9.0, although citrate buffer pH 6.0 can be used as an alternative .

• Positive Control: Human pancreas cancer tissue has been validated as a positive control for IHC applications .

• Optimization: It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as the optimal dilution may be sample-dependent .

How can researchers ensure the stability and activity of recombinant Tarsius syrichta COX6C during storage and experimental procedures?

To ensure the stability and activity of recombinant Tarsius syrichta COX6C:

• Storage Conditions:

  • Store the recombinant protein at -20°C for regular storage, or at -80°C for extended storage .

  • The shelf life of the liquid form is typically 6 months at -20°C/-80°C, while the lyophilized form can be stable for up to 12 months at -20°C/-80°C .

• Buffer Composition:

  • The recombinant protein is optimally stored in a Tris-based buffer with 50% glycerol .

  • This buffer composition has been specifically optimized for maintaining the stability of this particular protein.

• Handling Precautions:

  • Avoid repeated freeze-thaw cycles, as these can lead to protein degradation and loss of activity .

  • For working with the protein over short periods, store aliquots at 4°C for up to one week .

• Quality Control:

  • Verify protein integrity by SDS-PAGE before experimental use, looking for a single band at the expected molecular weight.

  • Consider functional assays specific to cytochrome c oxidase activity to confirm that the recombinant protein retains its functional properties.

What genetic analysis approaches can be used to study COX6C variation across different populations of Tarsius syrichta?

Based on conservation genetics studies of Tarsius syrichta, several approaches can be adapted to study COX6C variation across different populations:

• PCR Amplification and Sequencing:

  • Design primers specific to the COX6C gene region, similar to those used for other mitochondrial genes in Tarsius syrichta, such as ND2 and CytB .

  • Example primer design strategy could follow the pattern used for CytB markers: TarsCytB.F1, 5′–CACATCTGCCGAGACGTAAA–3′ and TarsCytB.R1, 5′–TGGGGTGGAGTGTTTAGAGG–3′ .

• Population Structure Analysis:

  • Utilize Bayesian clustering methods, as implemented in programs like Structure v2.3.3, to estimate population structure and infer the probabilities of individuals belonging to each of the estimated populations .

  • Run analyses under the correlated allele frequency model and the admixture ancestry model due to the absence of a priori knowledge of inter-locus relationships .

• Island Biogeography Considerations:

  • Account for the geographical distribution of Tarsius syrichta across the Mindanao PAIC (Philippine Archipelagic Island Complex) in the southern Philippines, including islands such as Samar, Leyte, Bohol, and Mindanao .

  • Consider the hypothesis that genetic diversity might be partitioned among individual islands, as suggested by previous studies .

• Conservation Implications:

  • Interpret COX6C variation in the context of conservation concerns, as Tarsius syrichta is currently threatened and endangered due to habitat loss and illegal pet trade .

  • Genetic diversity data can inform conservation strategies by identifying genetically distinct populations that may require specific protection measures.

How can recombinant Tarsius syrichta COX6C be used in comparative studies of primate mitochondrial function?

Recombinant Tarsius syrichta COX6C offers valuable opportunities for comparative studies of primate mitochondrial function, particularly given the unique evolutionary position of tarsiers within the primate lineage:

• Evolutionary Rate Analysis: Comparing COX6C sequences between Tarsius syrichta and other primates can reveal evolutionary rates and selective pressures on this component of the respiratory chain. The amino acid sequence provided (SSGALTKPQMRGLLAKRLRFHIVGAFAVSLGVAAFYKFAVAEPRKKAYADFYRNYDSMKDFEEMRKAGIFQSAK) can serve as a reference point for such comparisons .

• Functional Assays: Recombinant proteins from different primate species, including Tarsius syrichta COX6C, can be incorporated into functional assays to assess potential differences in cytochrome c oxidase activity, electron transfer efficiency, or assembly properties.

• Structure-Function Relationships: By comparing the structural features of COX6C across primates, researchers can identify conserved domains that are likely essential for function versus variable regions that may contribute to species-specific adaptations.

• Ecological Adaptations: Given the specific habitat requirements of Tarsius syrichta in tropical rainforests of the Philippines , comparative studies might reveal adaptations in mitochondrial proteins related to the species' unique ecological niche, nocturnal lifestyle, or metabolic requirements.

What can microsatellite analysis tell us about the genetic diversity of Tarsius syrichta populations and its implications for molecular studies?

Microsatellite analysis provides valuable insights into the genetic diversity of Tarsius syrichta populations, which has important implications for molecular studies:

• Population Structure: Microsatellite analysis of Tarsius syrichta across 17 localities in the southern Philippines has revealed patterns of genetic diversity that may be partitioned among individual islands . This population structure information is crucial for sampling strategies in molecular studies to ensure representation of genetic diversity.

• Genetic Variability Assessment: Researchers have used nine microsatellite markers (T5, T6, T22, T34, T35, T43, T69, T42, T50) to assess genetic variability within and between Tarsius syrichta populations . This baseline data on genetic diversity can inform expectations about variability in nuclear and mitochondrial genes, including those encoding components of the respiratory chain like COX6C.

• Admixture Analysis: Using Bayesian clustering methods, researchers can identify individuals with admixed ancestry (q values between 0.10 and 0.90) . This information on population admixture can help interpret patterns of molecular variation in genes of interest.

• Conservation Genetics Implications: The threatened status of Tarsius syrichta makes genetic diversity data particularly valuable for conservation planning. Molecular studies should consider how their sampling and analysis might contribute to conservation efforts for this endangered species.

What are the emerging research trends involving Cytochrome c oxidase subunit 6C in comparative primate studies?

Emerging research trends involving Cytochrome c oxidase subunit 6C in comparative primate studies include:

• Integrative Omics Approaches: Combining proteomic, transcriptomic, and metabolomic analyses to understand the role of COX6C within the broader context of mitochondrial function and cellular metabolism across primate species.

• Ecological Energetics: Investigating how variations in COX6C and other components of the respiratory chain might correlate with differences in energy requirements, habitat use, and activity patterns among primates, including specialized species like Tarsius syrichta.

• Conservation Physiology: Using molecular markers, including mitochondrial genes like COX6C, to assess population health and adaptive potential in endangered primates like Tarsius syrichta, which faces significant threats from deforestation and illegal pet trade .

• Evolutionary Medicine: Exploring how primate-specific variations in respiratory chain components like COX6C might inform our understanding of human mitochondrial disorders and aging-related diseases.

How can researchers contribute to both scientific advancement and conservation efforts for Tarsius syrichta through molecular studies?

Researchers can contribute to both scientific advancement and conservation efforts for Tarsius syrichta through molecular studies by:

• Ethical Sampling Protocols: Developing and adhering to non-invasive sampling protocols that minimize impact on wild populations while still obtaining valuable genetic material for molecular studies.

• Habitat-Specific Adaptations: Investigating potential adaptations in mitochondrial genes, including COX6C, that might be related to the species' specific habitat requirements in tropical rainforests of the Philippines .

• Genetic Health Monitoring: Using molecular markers to monitor genetic diversity and inbreeding in remaining wild populations, providing data that can inform conservation management decisions.

• Public Awareness: Communicating research findings in ways that highlight the unique evolutionary position of Tarsius syrichta and its ecological importance, thereby raising awareness about conservation needs.

• Collaborative Conservation Research: Engaging with conservation organizations and local communities in the Philippines to ensure that molecular research directly contributes to conservation action plans for this threatened species, which has suffered from hunting, trapping, and habitat loss .