Recombinant Aotus azarae Cytochrome b-c1 complex subunit Rieske, mitochondrial (UQCRFS1)

What is UQCRFS1 and what is its role in cellular metabolism?

UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1) is a critical component of the mitochondrial respiratory chain Complex III. The protein contains an iron-sulfur cluster that functions as an electron transfer component in oxidative phosphorylation. In Aotus azarae (Southern owl monkey), this protein plays an essential role in cellular energy production by facilitating electron transfer from ubiquinol to cytochrome c during ATP synthesis. The protein is anchored to the inner mitochondrial membrane and contains a characteristic [2Fe-2S] Rieske domain that is highly conserved across species .

How does Aotus azarae UQCRFS1 compare to homologs in other primates?

Comparative analysis of UQCRFS1 across primate species reveals high conservation of functional domains, particularly in the iron-sulfur cluster binding region. Phylogenetic studies indicate that Aotus azarae UQCRFS1 shares significant sequence homology with other New World monkeys, especially within the Platyrrhini clade. The protein shows approximately 95-98% sequence identity with other primates such as Saimiri sciureus (squirrel monkey) and higher primates. These similarities reflect the evolutionary importance of maintaining functional integrity of mitochondrial respiratory components across primate evolution .

What are the optimal conditions for expression and purification of recombinant Aotus azarae UQCRFS1?

For optimal expression of recombinant Aotus azarae UQCRFS1:

The recombinant protein typically requires denaturation and refolding protocols to ensure proper formation of the iron-sulfur cluster. Addition of iron and sulfur donors during the refolding process significantly improves the yield of functionally active protein .

What methods are effective for analyzing UQCRFS1 enzymatic activity in vitro?

Enzymatic activity of recombinant UQCRFS1 can be assessed through several complementary approaches:

• Spectrophotometric assays: Monitoring the reduction of cytochrome c at 550 nm in the presence of ubiquinol and purified UQCRFS1.

• Oxygen consumption measurements: Using oxygen electrodes to measure the rate of oxygen consumption in reconstituted systems.

• ROS production assessment: Using DCFH-DA fluorescent probe to measure reactive oxygen species generation, as UQCRFS1 function impacts ROS levels.

• Iron-sulfur cluster integrity analysis: Using electron paramagnetic resonance (EPR) spectroscopy to confirm proper assembly of the [2Fe-2S] cluster.

Researchers should note that active UQCRFS1 requires proper incorporation of the iron-sulfur cluster, and activity assays should include appropriate controls to account for background electron transfer reactions .

How can researchers effectively use Aotus azarae UQCRFS1 in comparative mitochondrial studies?

To effectively use Aotus azarae UQCRFS1 in comparative mitochondrial studies:

• Standardize expression systems: Express recombinant UQCRFS1 from multiple primate species using identical expression vectors and conditions to minimize methodological variability.

• Employ structural biology approaches: Use X-ray crystallography or cryo-EM to compare structural features across species.

• Conduct functional reconstitution experiments: Incorporate the recombinant protein into liposomes or isolated mitochondrial membranes depleted of endogenous UQCRFS1 to assess functional complementation.

• Perform evolutionary rate analysis: Compare substitution rates and selection pressures across primate lineages using appropriate molecular evolution models.

When conducting these studies, it's essential to maintain consistent biochemical conditions across all species being compared to ensure that observed differences are biological rather than methodological in nature .

How does UQCRFS1 function contribute to Aotus azarae's adaptation to nocturnal lifestyle?

Aotus azarae (Southern owl monkey) has evolved adaptations for nocturnal and cathemeral activity patterns. Research suggests that mitochondrial proteins, including UQCRFS1, may play roles in these adaptations:

• Metabolic rate regulation: UQCRFS1 efficiency may contribute to the specialized energy metabolism needed for nocturnal activity cycles.

• Oxidative stress management: Night monkeys may have evolved specific functional properties in their electron transport chain components to manage oxidative stress during shifting activity patterns.

• Tissue-specific expression patterns: UQCRFS1 expression profiles in retinal and neural tissues may differ from diurnal primates, supporting visual adaptation to low-light conditions.

What role might UQCRFS1 play in Aotus azarae's susceptibility to human pathogens?

Aotus species are important models for studying human diseases, particularly malaria. The potential role of UQCRFS1 in pathogen susceptibility may involve:

• Mitochondrial stress response: During infection, UQCRFS1 function may influence how cells respond to pathogen-induced mitochondrial stress.

• ROS-mediated defense mechanisms: UQCRFS1's role in electron transport affects ROS production, which plays roles in antimicrobial defense.

• Energy provision during immune activation: Efficient UQCRFS1 function may support the increased energy demands of immune response.

Research involving herpesvirus infection in Aotus azarae has demonstrated susceptibility to human pathogens, suggesting shared molecular mechanisms across primate species. Studies examining UQCRFS1 expression and function during experimental infections could reveal its role in host-pathogen interactions. This knowledge could inform the development of improved disease models and potential therapeutic targets .

How can UQCRFS1 knockdown experiments enhance our understanding of mitochondrial disease models?

UQCRFS1 knockdown experiments provide valuable insights into mitochondrial dysfunction:

These findings from human cell studies demonstrate that UQCRFS1 knockdown affects multiple cellular processes. Applying similar approaches to Aotus cell models could reveal species-specific responses to mitochondrial dysfunction and provide comparative insights into primate mitochondrial biology. Such experiments require careful optimization of siRNA design specific to the Aotus azarae UQCRFS1 sequence to ensure effective and specific knockdown .

How has UQCRFS1 evolved within the Aotus genus and how does this inform primate phylogenetics?

UQCRFS1 sequence analysis contributes to understanding Aotus phylogenetics:

• Conservation patterns: The coding regions of UQCRFS1 show high conservation across Aotus species, reflecting functional constraints on this essential mitochondrial protein.

• Phylogenetic signal: Comparative analysis of UQCRFS1 sequences supports the grouping of A. nancymaae and A. azarae (red-necked species) as sister taxa, with A. vociferans and A. trivirgatus (gray-necked species) being more basal in the phylogeny.

• Selection pressures: Analysis of selection patterns shows predominant purifying selection acting on UQCRFS1, though some amino acid sites may have experienced positive selection, particularly in regions not directly involved in electron transport.

When integrated with broader phylogenetic analyses using multiple genetic markers, UQCRFS1 data contributes to resolving relationships within the Aotus genus. This is particularly valuable given the historical challenges in establishing clear taxonomic boundaries among owl monkey species due to morphological similarities .

What methods are most effective for using UQCRFS1 as a molecular marker in primate evolutionary studies?

To effectively use UQCRFS1 as a molecular marker in primate evolutionary studies:

• Complete gene sequencing: Analyze both coding and non-coding regions (introns) of the UQCRFS1 gene to capture different evolutionary rates.

• Multi-locus approach: Combine UQCRFS1 with other mitochondrial and nuclear markers to increase phylogenetic resolution and account for gene tree/species tree discordance.

• Appropriate evolutionary models: Apply codon-based models that account for different selection pressures at different protein sites.

• Population-level sampling: Include multiple individuals per species to account for intraspecific variation.

• Comparative analysis with other Complex III components: Analyze patterns of co-evolution with functionally related proteins.

How can Aotus azarae UQCRFS1 inform research on human mitochondrial disorders?

Aotus azarae UQCRFS1 offers valuable comparative insights for human mitochondrial disorder research:

• Structural comparisons: Detailed structural analysis of Aotus UQCRFS1 compared to human homologs (which share approximately 90% sequence identity) can identify conserved regions critical for function versus regions that tolerate variation.

• Functional complementation studies: Testing whether Aotus UQCRFS1 can functionally replace defective human UQCRFS1 in cell models could reveal species-specific functional properties.

• Adaptive differences: Identifying amino acid changes specific to the Aotus lineage may reveal adaptations that affect mitochondrial efficiency or stress tolerance.

These approaches could provide insights into the pathogenic mechanisms of human UQCRFS1 mutations and potentially identify novel therapeutic strategies for mitochondrial disorders. The high sequence conservation between human and Aotus UQCRFS1 makes this comparison particularly relevant for biomedical applications .

What evidence exists for UQCRFS1's role in oxidative stress and apoptotic pathways?

Experimental evidence demonstrates UQCRFS1's significant involvement in oxidative stress and apoptotic pathways:

• ROS production: UQCRFS1 knockdown experiments show markedly enhanced production of reactive oxygen species (ROS), as measured by DCFH-DA fluorescent probes.

• DNA damage response: UQCRFS1 depletion upregulates DNA damage response genes including ATM and ATR, while downregulating CHK1 and CHK2.

• Apoptotic signaling: Western blot analyses reveal that UQCRFS1 knockdown significantly decreases expression of anti-apoptotic proteins including Bcl-2 and increases the percentage of apoptotic cells.

• Cell cycle effects: UQCRFS1 depletion causes G1 phase arrest and decreased expression of cell cycle regulatory proteins (cyclin D1, CDK2, CDK4).

These findings suggest that UQCRFS1 modulates cellular homeostasis through its effects on electron transport, ROS generation, and subsequent signaling pathways. Comparative studies between human and Aotus UQCRFS1 could reveal whether these mechanisms are conserved across primates .

How might comparative analysis of UQCRFS1 across primate species contribute to cancer research?

Comparative analysis of UQCRFS1 across primates offers potential insights for cancer research:

• Expression pattern conservation: Studying whether UQCRFS1 overexpression patterns in certain cancers are consistent across primate species could identify conserved regulatory mechanisms.

• Functional domain analysis: Identifying primate-specific variations in functional domains could reveal how subtle structural differences affect cancer-relevant functions.

• Signaling pathway interactions: Investigating whether UQCRFS1's interactions with cancer-relevant pathways (such as AKT/mTOR) are conserved across primates could highlight essential versus species-specific mechanisms.

Studies have shown that in human cancer models, UQCRFS1 knockdown inhibits the AKT/mTOR pathway, reduces proliferation, induces apoptosis, and increases ROS production. Comparative analysis of these phenotypes in Aotus cell models could provide evolutionary context for these cancer-relevant functions. Such research could potentially identify novel therapeutic targets or improve the predictive value of primate cancer models .

What are the main challenges in producing functional recombinant Aotus azarae UQCRFS1 and how can they be overcome?

Production of functional recombinant Aotus azarae UQCRFS1 presents several challenges:

Researchers have reported success using E. coli expression systems with controlled induction conditions followed by affinity purification and careful refolding protocols. The addition of 6% trehalose in storage buffers significantly improves protein stability during freeze-thaw cycles .

What experimental controls are essential when studying the effects of recombinant UQCRFS1 in cellular systems?

When studying recombinant UQCRFS1 effects in cellular systems, the following controls are essential:

• Expression level verification: Western blot analysis to confirm that the recombinant protein is expressed at levels comparable to endogenous UQCRFS1.

• Functional activity control: Measurement of Complex III activity to verify that the recombinant protein is functionally active.

• Subcellular localization verification: Immunofluorescence or subcellular fractionation to confirm proper mitochondrial targeting.

• Empty vector control: Cells transfected with expression vector lacking the UQCRFS1 insert.

• Inactive mutant control: Cells expressing a non-functional UQCRFS1 mutant (e.g., with mutations in the iron-sulfur cluster binding site).

• Species-matched control: When comparing across species, ensure matching expression levels and cellular backgrounds.

These controls help distinguish specific effects of UQCRFS1 from non-specific effects of the expression system or experimental manipulation .

How can researchers accurately assess interactions between UQCRFS1 and other components of the respiratory chain?

To accurately assess interactions between UQCRFS1 and other respiratory chain components:

• Co-immunoprecipitation studies: Using antibodies against UQCRFS1 or other complex III components to identify physical interactions.

• Blue native PAGE: To analyze intact respiratory complexes and supercomplexes containing UQCRFS1.

• Proximity labeling techniques: BioID or APEX2 fusion proteins to identify proteins in close proximity to UQCRFS1 in living cells.

• Crosslinking mass spectrometry: To identify specific interaction interfaces between UQCRFS1 and other proteins.

• FRET/BRET analysis: To study dynamic interactions using fluorescent or bioluminescent fusion proteins.

• Reconstitution experiments: Using purified components to rebuild partial or complete respiratory complexes in vitro.

When conducting these studies with recombinant Aotus azarae UQCRFS1, researchers should verify that any tags or modifications do not interfere with protein interactions, and should include appropriate controls to distinguish specific from non-specific interactions .