Recombinant Saimiri sciureus Cytochrome b-c1 complex subunit Rieske, mitochondrial (UQCRFS1)

What is UQCRFS1 and what is its role in mitochondrial function?

UQCRFS1 (Ubiquinol-cytochrome c reductase Rieske iron-sulfur protein) serves as one of the catalytic subunits of the cytochrome bc1 complex (complex III) in the mitochondrial respiratory chain. This protein is critically involved in electron transfer processes occurring at the inner mitochondrial membrane. The Rieske iron-sulfur protein contains a 2Fe-2S cluster that participates in the electron transport chain, facilitating oxidative phosphorylation and cellular energy production. UQCRFS1 is encoded by nuclear DNA and must be transported from the cytosol into mitochondria via a cleavable N-terminal presequence, where it subsequently incorporates the iron-sulfur cluster and assembles with other subunits to form the functional cytochrome bc1 complex .

How does Saimiri sciureus UQCRFS1 compare to human UQCRFS1?

Table 1: Comparative Analysis of Saimiri sciureus and Human UQCRFS1

The high degree of conservation between squirrel monkey and human UQCRFS1, particularly in the iron-sulfur cluster binding domains, suggests that findings from studies using the recombinant Saimiri sciureus protein may offer translational value for human health research, especially in mitochondrial dysfunction studies.

How can recombinant UQCRFS1 be used to study mitochondrial complex assembly?

Recombinant UQCRFS1 offers a powerful tool for investigating the stepwise assembly of mitochondrial complex III. Researchers can employ various methodological approaches:

• In vitro reconstitution assays: Using purified recombinant UQCRFS1 along with other complex III subunits to study assembly kinetics and intermediate complexes. This technique involves incubating the recombinant protein with isolated mitochondrial membranes depleted of native UQCRFS1, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) to analyze complex formation.

• Crosslinking studies: Chemical crosslinking of recombinant UQCRFS1 with other complex III components allows identification of proximity relationships and interaction sites. When combined with mass spectrometry, this approach can map the three-dimensional arrangement of subunits within the complex.

• Mutational analysis: Systematic mutation of conserved residues in recombinant UQCRFS1 can reveal critical regions required for complex assembly. Studies with bacterial expression systems have demonstrated that the Rieske protein can independently assemble an iron-sulfur cluster and associate with membranes even in the absence of other complex III components, suggesting a distinct membrane attachment mechanism .

• Import assays: Radiolabeled recombinant UQCRFS1 can be used in mitochondrial import assays to study the mechanisms of protein translocation, processing by mitochondrial proteases, and incorporation into the respiratory chain complexes.

These approaches contribute to understanding the hierarchical assembly process of complex III and the specific role of UQCRFS1 in maintaining structural integrity and electron transport functionality.

What is the significance of UQCRFS1 as a biomarker in cancer research?

Recent studies have identified UQCRFS1 as a potential prognostic biomarker in several cancer types. In ovarian cancer, high expression of UQCRFS1 is associated with poor prognosis and contributes to tumor progression through multiple mechanisms . The significance of UQCRFS1 in cancer research involves:

• Altered metabolic programming: High UQCRFS1 expression positively correlates with oxidative phosphorylation (OXPHOS) gene signatures, suggesting its role in cancer metabolic reprogramming. This correlation indicates that UQCRFS1 may be critical for maintaining OXPHOS function in cancer cells .

• Cell cycle regulation: UQCRFS1 expression positively correlates with cell cycle progression genes (CDK2, CDK4, CCNE1) and negatively correlates with apoptosis genes (ADD1, BAX, FAS). Knockdown of UQCRFS1 in ovarian cancer cell lines resulted in G1 phase cell cycle arrest and decreased expression of cyclin D1, CDK2, and CDK4 .

• Redox homeostasis: UQCRFS1 knockdown increased reactive oxygen species (ROS) production and upregulated DNA damage response genes like ATM and ATR. This suggests that UQCRFS1 helps maintain redox balance in cancer cells, potentially contributing to genomic stability and treatment resistance .

• Signaling pathway modulation: UQCRFS1 appears to influence the AKT/mTOR pathway, with knockdown experiments showing inhibition of this critical oncogenic signaling axis . This connection provides insight into how mitochondrial respiratory chain components may impact canonical cancer signaling networks.

Researchers can utilize recombinant UQCRFS1 to develop standardized assays for detecting its overexpression in clinical samples, potentially aiding in cancer diagnosis, prognosis, and treatment selection.

What are the optimal conditions for expressing recombinant Saimiri sciureus UQCRFS1?

Table 2: Optimization Parameters for Recombinant UQCRFS1 Expression

For optimal expression of functional recombinant UQCRFS1, it's crucial to consider the iron-sulfur cluster assembly. Research has shown that the unfused Rieske subunit expressed in E. coli can assemble a Rieske-like iron-sulfur cluster, though the EPR characteristics differ from those observed in the native cytochrome bc1 complex. Interestingly, when expressed in R. sphaeroides in the absence of cytochrome b and c1 subunits, the Rieske protein assembles a fully metalated iron-sulfur cluster with the diagnostic gy = 1.90 EPR signal and successfully integrates into the cytoplasmic membrane . This demonstrates that the protein possesses an intrinsic capacity for membrane attachment independent of other complex components.

What analytical techniques are most effective for assessing UQCRFS1 structure and function?

Multiple complementary techniques should be employed to comprehensively characterize recombinant UQCRFS1:

• Spectroscopic methods:

  • UV-visible spectroscopy: Monitors the characteristic absorption peaks of the iron-sulfur cluster (320-500 nm range).

  • Electron Paramagnetic Resonance (EPR): Critical for assessing the integrity and environment of the 2Fe-2S cluster. Native Rieske proteins typically exhibit a rhombic signal with characteristic g-values (gy = 1.90) .

  • Circular Dichroism (CD): Evaluates secondary structure content and thermal stability of the recombinant protein.

• Functional assays:

  • Electron transfer activity: Measure the rate of cytochrome c reduction using decylubiquinol as electron donor.

  • Oxygen consumption: Assess integration into respiratory chain complexes using a Clark-type oxygen electrode.

  • Membrane binding assays: Ultracentrifugation-based fractionation to determine membrane association efficiency.

• Structural characterization:

  • Limited proteolysis: Identifies flexible regions and domain boundaries.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps solvent-exposed regions and protein dynamics.

  • Crosslinking coupled with mass spectrometry: Determines intramolecular and intermolecular interaction sites.

• Iron-sulfur cluster analysis:

  • Inductively coupled plasma mass spectrometry (ICP-MS): Quantifies iron content.

  • Mössbauer spectroscopy: Characterizes the oxidation state and environment of iron atoms.

  • Resonance Raman spectroscopy: Provides information about Fe-S bond vibrations.

These complementary approaches provide a comprehensive assessment of both structural integrity and functional competence of the recombinant protein, ensuring its suitability for downstream applications in mitochondrial research or drug discovery.

How can researchers effectively evaluate UQCRFS1 interactions with other complex III components?

Investigating UQCRFS1 interactions with other complex III components requires a multi-faceted approach:

• Co-immunoprecipitation (Co-IP): Using antibodies against recombinant UQCRFS1 to pull down interacting partners from mitochondrial lysates. This approach can be enhanced with chemical crosslinking to capture transient interactions.

• Surface Plasmon Resonance (SPR): Immobilize recombinant UQCRFS1 on a sensor chip and flow other purified complex III components to measure binding kinetics and affinity constants.

• Isothermal Titration Calorimetry (ITC): Directly measure thermodynamic parameters of UQCRFS1 interactions with other subunits, providing insights into binding stoichiometry, enthalpy, and entropy changes.

• Yeast two-hybrid and split-luciferase complementation assays: For mapping specific interaction domains between UQCRFS1 and other complex III proteins.

• Reconstitution experiments: Sequential addition of purified components, including recombinant UQCRFS1, to liposomes or nanodiscs, followed by activity measurements to determine the minimal components required for electron transfer.

• Microscale Thermophoresis (MST): A solution-based technique requiring small sample amounts to measure interactions between fluorescently labeled UQCRFS1 and unlabeled binding partners.

• Cryo-EM analysis: Reconstitution of complex III with and without recombinant UQCRFS1 to visualize structural changes and determine the protein's position within the complex.

These methodologies provide complementary data on the specificity, strength, and structural basis of UQCRFS1 interactions within the respiratory chain complex, advancing our understanding of mitochondrial respiratory complex assembly.

How does recombinant UQCRFS1 expression relate to its functional activity in mitochondrial studies?

When working with recombinant UQCRFS1, researchers must carefully assess the relationship between expression levels and functional activity. Studies have demonstrated that simply achieving high expression does not guarantee functional incorporation of the iron-sulfur cluster. For example, fusion proteins like maltose binding protein (MBP)-UQCRFS1 may express well but fail to incorporate an EPR-detectable iron-sulfur cluster .

Key considerations for correlating expression with activity include:

• Iron-sulfur cluster incorporation: The presence of a Rieske-like iron-sulfur cluster can be assessed by EPR spectroscopy. The unfused Rieske subunit expressed in E. coli assembles an iron-sulfur cluster but with EPR characteristics that differ from the normal rhombic signal observed in the native cytochrome bc1 complex . Complete functional assessment requires both spectroscopic confirmation of cluster assembly and enzymatic activity measurements.

• Membrane localization: Proper subcellular localization is critical for function. When expressed in heterologous systems, UQCRFS1 demonstrates an intrinsic ability to associate with membranes even in the absence of other complex III components. In Rhodobacter sphaeroides lacking other bc1 complex subunits, the fully metalated Rieske protein with the diagnostic gy = 1.90 EPR signal localizes to the cytoplasmic membrane . This suggests that membrane attachment mechanisms are partially independent of protein-protein interactions within the complex.

• Correlation with physiological effects: In cellular studies, UQCRFS1 expression levels correlate with multiple physiological parameters. High expression positively correlates with oxidative phosphorylation and cell cycle progression genes, while negatively correlating with apoptosis and DNA damage response genes . These correlations provide functional readouts for assessing recombinant protein activity in cellular contexts.

• N-terminal processing: The N-terminal region is critical for both membrane attachment and proper folding. Expression constructs lacking the amino-terminal hydrophobic anchor region fail to assemble the iron-sulfur cluster when expressed in E. coli , highlighting the importance of this domain for functional protein production.

What are the comparative advantages of UQCRFS1 studies across different species models?

Table 3: Comparative Analysis of UQCRFS1 Across Species Models

Researchers should leverage the specific advantages of each model system based on their research questions. For fundamental mechanistic studies of iron-sulfur cluster assembly, bacterial systems offer experimental tractability. For investigations related to mitochondrial import and processing, yeast models excel. For disease-relevant applications and biomarker development, primate and human systems provide the most translational value.

How do UQCRFS1 mutations impact mitochondrial function and disease pathology?

UQCRFS1 mutations have profound implications for mitochondrial function and are associated with several pathological conditions:

• Mitochondrial complex III deficiency: Genetic deletion of UQCRFS1 causes complex III deficiency, resulting in cardiomyopathy and alopecia totalis . This underscores the protein's essential role in respiratory chain function and cellular energy production.

• Cancer progression: UQCRFS1 overexpression has been documented in various cancers, including ovarian , breast, gastric, and melanoma . The mechanistic connections between UQCRFS1 and cancer progression involve:

  • Promotion of cell proliferation through cell cycle regulation

  • Inhibition of apoptosis

  • Modulation of reactive oxygen species (ROS) levels

  • Influence on DNA damage response pathways

  • Regulation of the AKT/mTOR signaling pathway

• Reactive oxygen species (ROS) production: UQCRFS1 plays a critical role in regulating ROS production. Knockdown of UQCRFS1 significantly increases intracellular ROS levels, leading to oxidative stress and activation of DNA damage response pathways . This suggests that proper UQCRFS1 function is essential for maintaining redox homeostasis.

• Metabolic reprogramming: UQCRFS1 expression positively correlates with oxidative phosphorylation genes, suggesting its role in metabolic regulation . Alterations in UQCRFS1 may contribute to the metabolic reprogramming observed in cancer and other diseases.

• Therapeutic targeting: The involvement of UQCRFS1 in cancer progression suggests its potential as a therapeutic target. Inhibition of UQCRFS1 could potentially reduce cancer cell proliferation, induce cell cycle arrest, promote apoptosis, and inhibit the AKT/mTOR pathway .

These findings highlight the multifaceted roles of UQCRFS1 in cellular physiology and disease pathology, making it an important subject for both basic research and translational medicine.

What are common challenges in recombinant UQCRFS1 expression and how can they be addressed?

Table 4: Troubleshooting Guide for Recombinant UQCRFS1 Expression

Research has shown that expression system selection significantly impacts recombinant UQCRFS1 functionality. While the protein can be expressed in E. coli, the resulting product may contain an iron-sulfur cluster with altered EPR characteristics. In contrast, expression in Rhodobacter sphaeroides appears to yield a more native-like protein with the characteristic EPR signal, even in the absence of other complex III components . This suggests that prokaryotic expression systems can produce functional protein, but careful optimization and characterization are essential.

How can researchers resolve contradictory data in UQCRFS1 functional studies?

When confronted with contradictory results in UQCRFS1 research, systematic analysis and reconciliation approaches are essential:

• Expression system variations: Different expression systems yield UQCRFS1 proteins with varying functionality. For example, studies have shown that unfused Rieske protein expressed in E. coli assembles an iron-sulfur cluster with different EPR characteristics compared to the native complex, while MBP-fusion constructs fail to incorporate detectable clusters . When comparing results across studies, researchers should thoroughly document and consider the expression system's influence.

• Post-translational modification disparities: Recombinant proteins often lack native post-translational modifications that could be critical for function. Experimental approaches should include:

  • Parallel testing of recombinant and native proteins

  • Mass spectrometry analysis to identify missing modifications

  • Complementary in vitro and in vivo functional assays

• Iron-sulfur cluster integrity validation: Contradictory functional data may stem from variations in iron-sulfur cluster incorporation. Multiple independent methods should verify cluster integrity:

  • EPR spectroscopy

  • Iron quantification

  • Activity assays

  • CD spectroscopy for structural assessment

• Methodological reconciliation: When studies report contradictory findings on UQCRFS1 function in cancer, rigorous analysis is required:

  • Cell type differences: UQCRFS1 may have context-dependent effects across cancer types

  • Knockdown efficiency variations: Partial vs. complete UQCRFS1 depletion may yield different phenotypes

  • Timepoint discrepancies: Acute vs. chronic UQCRFS1 alterations may trigger different cellular responses

• Integrated data analysis: Comprehensive meta-analysis of published data combined with orthogonal experimental approaches can help resolve contradictions. For instance, high-throughput transcriptomic and proteomic studies can validate or refute hypothesized mechanisms of UQCRFS1 in cancer progression.

By systematically addressing these potential sources of contradiction, researchers can develop a more coherent understanding of UQCRFS1 biology across experimental systems.

What quality control measures are essential for ensuring reproducible results with recombinant UQCRFS1?

Rigorous quality control is crucial for generating reliable and reproducible data with recombinant UQCRFS1:

• Protein purity assessment:

  • SDS-PAGE with Coomassie staining (≥95% purity)

  • Western blot with specific antibodies

  • Mass spectrometry to confirm protein identity and detect modifications

  • Size-exclusion chromatography to evaluate aggregation state

• Iron-sulfur cluster integrity verification:

  • UV-visible spectroscopy for characteristic absorbance peaks

  • EPR spectroscopy to confirm proper cluster environment (rhombic signal with gy = 1.90 for native-like protein)

  • Iron:protein ratio determination (typically 2:1 for fully loaded protein)

  • Enzymatic activity assays (cytochrome c reduction)

• Structural validation:

  • Circular dichroism to confirm secondary structure integrity

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to verify proper folding

  • Dynamic light scattering to detect aggregation

• Functional characterization:

  • Membrane binding efficiency

  • Electron transfer activity

  • Interaction with known binding partners

  • Response to inhibitors and oxidative stress

• Batch consistency monitoring:

  • Maintain detailed expression and purification records

  • Implement reference standard comparisons between batches

  • Perform lot-to-lot functional comparisons

  • Document storage conditions and freeze-thaw cycles

• Documentation and reporting standards:

  • Comprehensive methods sections in publications

  • Deposition of detailed protocols in repositories

  • Sharing of raw data when possible

  • Clear description of expression constructs, systems, and purification methods

Implementation of these quality control measures ensures that experimental outcomes reflect genuine biological phenomena rather than artifacts arising from protein preparation variability. Given the sensitivity of iron-sulfur proteins to oxidation and the complexity of their folding and assembly, stringent quality control is particularly critical for UQCRFS1 research.