Recombinant Macaca fascicularis NADH dehydrogenase [ubiquinone] 1 subunit C2 (NDUFC2)

What is NDUFC2 and what is its primary function in mitochondria?

NDUFC2 (NADH dehydrogenase [ubiquinone] 1 subunit C2) is a critical component of Complex I (NADH:ubiquinone oxidoreductase) within the mitochondrial oxidative phosphorylation (OXPHOS) system. It functions primarily in the inner mitochondrial membrane where it plays a crucial role in facilitating proper Complex I assembly and activity . Complex I represents the largest complex of the mitochondrial OXPHOS system and is considered the major site of reactive oxygen species (ROS) generation within mitochondria .

The protein is also known as CI-B14.5b (Complex I-B14.5b) and is strategically positioned in the membrane arm of Complex I, which enables its interactions with other subunits and facilitates electron transport across the mitochondrial membrane . Functionally, NDUFC2 contributes to energy production by helping maintain the electron transport chain that generates the proton gradient necessary for ATP synthesis.

What experimental systems are optimal for studying recombinant NDUFC2 from Macaca fascicularis?

The following experimental systems have proven effective for studying Macaca fascicularis NDUFC2:

• Cell Culture Systems:

  • Primary fibroblast cultures from macaque skin samples provide a physiologically relevant system for studying native NDUFC2 function

  • Heterologous expression systems using mammalian cell lines can be used for recombinant protein production

• Animal Models:

  • Knock-out rat models (such as the SHRSR_KO model) have been successfully used to study the effects of NDUFC2 deficiency on mitochondrial function and structure

  • Cynomolgus macaques themselves serve as important non-human primate models, particularly those from Mauritius which have a well-documented genetic background

• Biochemical Approaches:

  • Recombinant protein expression and purification for structural and functional studies

  • ELISA-based assays for quantification and interaction studies

• Genetic Systems:

  • Gene silencing approaches (siRNA/shRNA) can be used to study the effects of NDUFC2 reduction in cellular models

  • Genome editing techniques can be applied to introduce specific mutations identified in evolutionary or disease-associated studies

For optimal results, researchers should consider using multiple complementary approaches, as each system offers different advantages for investigating specific aspects of NDUFC2 biology.

What are the known functional domains of the NDUFC2 protein?

While the search results don't provide a comprehensive domain mapping of NDUFC2, several functional features can be inferred:

• Membrane-Associated Region: NDUFC2 is located in the membrane arm of Complex I, suggesting the presence of hydrophobic regions that facilitate membrane association

• Interaction Sites: The protein contains specific amino acids involved in interactions with other Complex I subunits, particularly:

  • Cysteine 39 (C39), which appears to change in parallel with C330 of the mtDNA-encoded ND5 gene across species, suggesting a potential interaction site

  • Regions containing the charge-altering amino acid substitutions (Q78E, E114K, and K103E) that may mediate electrostatic interactions

• Conserved Regions: The 15 positively charged amino acids that are conserved across vertebrates likely play critical structural or functional roles

• Mitochondrial Targeting Signal: As a mitochondrial protein, NDUFC2 likely contains sequences directing its import into mitochondria and proper assembly into Complex I

Detailed structural studies combining crystallography, cryo-electron microscopy, and computational modeling would be needed to fully characterize these domains.

How does NDUFC2 deficiency affect mitochondrial structure and function?

NDUFC2 deficiency causes significant impairments in both mitochondrial function and structure, as demonstrated in studies using a heterozygous knockout rat model . The functional and structural alterations include:

Functional Impairments:

• Reduced Complex I assembly and activity

• Decreased mitochondrial membrane potential

• Increased production of reactive oxygen species (ROS)

• Impaired cellular energy production

Structural Alterations:
Mitochondrial ultrastructural damage was quantified using two key parameters :

• Percentage of mitochondrial area with intact cristae

• Inner mitochondrial membrane (IMM)/outer mitochondrial membrane (OMM) index

The morphological damage was categorized into three levels of severity :

These structural alterations were consistent with the functional impairment of Complex I and increased oxidative stress observed both in vitro and in vivo. Importantly, similar morpho-functional impairments were observed in peripheral blood mononuclear cells (PBMCs) from human subjects carrying genetic variants associated with decreased NDUFC2 expression .

What methodologies are optimal for assessing NDUFC2 function in experimental systems?

Several complementary methodologies can be employed to comprehensively assess NDUFC2 function:

• Complex I Assembly and Activity Assays:

  • Blue Native-PAGE followed by in-gel activity staining

  • Spectrophotometric assays measuring NADH oxidation rates

  • Polarographic measurements of oxygen consumption

• Mitochondrial Membrane Potential Assessment:

  • Fluorescent probes (e.g., JC-1, TMRM, Rhodamine 123)

  • Flow cytometry for quantitative analysis

• ROS Production Measurement:

  • Fluorescent probes (e.g., DCF-DA, MitoSOX Red)

  • Electron paramagnetic resonance (EPR) spectroscopy

• Mitochondrial Ultrastructure Analysis:

  • Transmission electron microscopy (TEM)

  • Quantification of mitochondrial area with intact cristae

  • Measurement of IMM/OMM index

• Gene Expression Analysis:

  • qRT-PCR for NDUFC2 mRNA quantification

  • Microarray analysis with probe-set standardization

  • RNA-Seq for comprehensive transcriptomic profiling

• Protein Expression and Interaction Studies:

  • Western blotting for protein level quantification

  • Co-immunoprecipitation for interaction studies

  • Proximity ligation assays for in situ interaction detection

• Functional Consequences Assessment:

  • ATP production assays

  • Cell viability and proliferation assays

  • Metabolic flux analysis

For rigorous research, a combination of these methodologies should be employed to provide multiple lines of evidence regarding NDUFC2 function in the experimental system of interest.

How does NDUFC2 interact with other subunits of Complex I?

NDUFC2 engages in critical interactions with both nuclear-encoded and mitochondrial DNA-encoded subunits of Complex I to ensure proper assembly and function. Key interaction features include:

• Co-localization with ND5: NDUFC2 co-localizes with the mtDNA-encoded ND5 subunit in sub-complex Iβ of Complex I .

• Cysteine-Mediated Interactions: Evidence suggests that C39 of NDUFC2 interacts with C330 of the mtDNA-encoded ND5 gene, as these residues change in parallel during evolution in both G. gallus and Drosophila . This suggests a potential disulfide bridge or other cysteine-mediated interaction.

• Charge-Based Interactions: NDUFC2 contains several charge-altering amino acid substitutions (Q78E, E114K, and K103E) that emerged in the Hominini lineage . These changes may facilitate electrostatic interactions with other subunits within Complex I.

• Evolutionary Co-variation: Interacting nDNA and mtDNA subunits would be expected to change in parallel during species radiation . This co-evolutionary pattern is observed between NDUFC2 and mtDNA-encoded subunits, supporting their functional interaction.

• Membrane Arm Positioning: As part of the membrane domain of Complex I, NDUFC2 is positioned to interact with other membrane-embedded subunits, particularly those involved in proton pumping.

These interactions highlight the importance of NDUFC2 in maintaining the structural integrity and functional capacity of Complex I, explaining why its deficiency leads to significant mitochondrial dysfunction.

What differences exist in NDUFC2 between Macaca fascicularis and other primate species?

NDUFC2 exhibits several evolutionary differences between Macaca fascicularis and other primate species, reflecting both genetic divergence and instances of introgression:

• Ancestral Introgression: Cynomolgus macaques (M. fascicularis) and rhesus macaques (M. mulatta) have a long history of ancestral introgression affecting nuclear genes like NDUFC2 . This has created complex patterns of genetic similarity and difference between these species.

• Population-Specific Variation: Isolated populations of M. fascicularis, such as those on Mauritius, have undergone genetic bottlenecks (estimated ~20 founding individuals) , potentially affecting the genetic diversity of NDUFC2 in these populations.

• Evolutionary Selection: NDUFC2 underwent positive selection at specific times in primate evolution, with certain amino acid changes occurring at:

  • The emergence of apes

  • Following the emergence of orangutan

• Mitonuclear Compatibility: In cases of mitochondrial DNA introgression between macaque species, nuclear genes like NDUFC2 show evidence of selection to maintain compatibility with the introgressed mitochondrial genome . For example:

  • In M. arctoides, which carries mitochondrial DNA closely related to M. mulatta but a nuclear genome more similar to sinica group macaques, gene flow between M. mulatta and M. arctoides was higher in genomic windows containing NDUFC2 and other N-interact genes

  • Similarly, M. fascicularis aurea, which carries introgressed mitochondria from an ancestor of sinica group macaques, shows evidence of selection on nuclear genes that interact with mitochondrial components

• Functional Constraints: Despite these evolutionary differences, NDUFC2 maintains its critical function in Complex I across primate species, suggesting strong functional constraints on certain regions of the protein.

These differences reflect the evolutionary history of macaques and demonstrate how mitonuclear interactions shape the genetic diversity of species and populations.

How do mitonuclear interactions involving NDUFC2 influence macaque species diversity and social structures?

Mitonuclear interactions, including those involving NDUFC2, have significantly shaped macaque evolution and social structures through several mechanisms:

• Female Philopatry and Genetic Variation:

  • In macaque societies with extreme female philopatry (females remain in their birth group), contrasting distributions of genetic variation in mitochondrial and nuclear genomes create variation in mitonuclear interactions

  • This pattern creates a unique selective landscape where mitonuclear compatibility becomes particularly important

• Natural Selection on Mitonuclear Interactions:

  • Analysis of genomic data sets from multiple macaque species revealed atypically long runs of homozygosity, low polymorphism, high differentiation, and/or rapid protein evolution associated with nuclear genes that interact with mitochondrial components (N-interact genes), including NDUFC2

  • These metrics suggest these genes were independently subject to atypically pervasive natural selection in multiple species

• Impact on Macaque Societies:
  The evidence suggests natural selection on mitonuclear interactions has influenced:

  • Species diversity: Contributing to the diversification of macaque species

  • Ecological breadth: Enabling adaptation to diverse environments

  • Female-biased adult sex ratio and demography: Reinforcing social structures with female philopatry

  • Sexual dimorphism: Potentially driving differences between males and females

  • Mitonuclear phylogenomics: Creating complex patterns of nuclear and mitochondrial genome relationships

• Mitochondrial Introgression and Nuclear Adaptation:

  • Several macaque species show evidence of mitochondrial DNA introgression from distantly related species

  • For example, M. arctoides carries mitochondrial DNA related to fascicularis group macaques but a nuclear genome more similar to sinica group macaques

  • In these cases, gene flow of N-interact genes like NDUFC2 shows patterns consistent with selection for mitonuclear compatibility

These findings demonstrate how the molecular interaction between nuclear-encoded NDUFC2 and mitochondrial components has influenced macaque evolution at multiple levels, from molecular function to social organization.

What molecular mechanisms connect NDUFC2 deficiency to increased disease susceptibility?

NDUFC2 deficiency contributes to disease susceptibility through several interconnected molecular mechanisms:

• Complex I Dysfunction:

  • Impaired assembly and activity of Complex I

  • Reduced ATP production

  • Increased electron leakage from the respiratory chain

• Elevated Oxidative Stress:

  • Increased production of reactive oxygen species (ROS)

  • Oxidative damage to proteins, lipids, and DNA

  • Activation of stress response pathways

• Mitochondrial Structural Damage:

  • Ultrastructural alterations characterized by damaged cristae

  • Abnormal inner mitochondrial membrane morphology

  • Impaired mitochondrial dynamics (fusion/fission)

• Tissue-Specific Effects:

  • In the heterozygous knock-out rat model, NDUFC2 deficiency led to renal damage followed by stroke when fed with a Japanese-style diet

  • Different tissues show varying sensitivity to NDUFC2 deficiency based on their metabolic demands

• Biomarker Potential:

  • Similar morpho-functional impairment observed in peripheral blood mononuclear cells (PBMCs) from healthy carriers with decreased NDUFC2 expression

  • Suggests NDUFC2 deficiency could serve as a biomarker for increased disease susceptibility

These mechanisms create a cellular environment prone to energy deficiency, oxidative damage, and impaired stress responses, ultimately increasing susceptibility to various diseases, particularly those affecting tissues with high energy demands like the brain, heart, and kidneys.

How can advanced complexome profiling techniques be applied to study NDUFC2-containing complexes?

Complexome profiling offers sophisticated approaches to study NDUFC2 incorporation into Complex I and other potential protein interactions:

• Methodological Workflow for Complexome Profiling:

  • Sample Preparation:

    • Isolation of intact mitochondria from relevant tissues or cell lines

    • Gentle solubilization using non-denaturing detergents to preserve protein complexes

  • Complex Separation:

    • Blue-native electrophoresis (BNE) or

    • Size-exclusion chromatography (SEC)

  • Fractionation and Mass Spectrometry:

    • Division of gel or chromatographic run into fractions

    • Bottom-up mass spectrometry (MS)-based proteomics analysis of each fraction

    • Peptide identification and protein quantification across fractions

• Data Analysis Approaches:

  • Correlation-Based Analysis:

    • Identification of proteins with elution profiles similar to NDUFC2

    • Clustering of proteins with similar migration patterns

  • Complex-Centric Approaches:

    • Automated workflows comparing abundance of known protein complexes

    • Utilization of curated protein complex databases as ground-truth

  • PERCOM (Protein Elution profile Reconnaissance through COMputational analysis):

    • "Protein-centric" strategy that systematically analyzes all detected proteins

    • Identification of proteins with altered elution profiles without database constraints

    • Particularly valuable for studying specialized complexes in specific tissues

• NDUFC2-Specific Applications:

  • Assembly Status Assessment:

    • Determination of whether NDUFC2 is properly incorporated into Complex I

    • Detection of subcomplexes or assembly intermediates containing NDUFC2

  • Novel Interaction Discovery:

    • Identification of previously unknown NDUFC2 interaction partners

    • Characterization of tissue-specific or condition-specific interactions

  • Comparative Studies:

    • Comparison of NDUFC2-containing complexes between different species

    • Analysis of complex alterations in disease models or patients with NDUFC2 variants

  • Structural Integrity Assessment:

    • Evaluation of how mutations or environmental factors affect Complex I integrity

    • Quantification of properly assembled vs. abnormal complexes

• Advantages Over Traditional Methods:

  • Captures native protein complexes

  • Provides comprehensive overview of all complexes simultaneously

  • Unbiased discovery of novel interactions

  • Detection of subtle changes in complex composition or abundance

This approach overcomes limitations of database-dependent methods, which often have "severe underrepresentation and poor coverage of specialized and cell-type specific complexes" , making it particularly valuable for studying NDUFC2 in specialized tissues like brain or heart.

What strategies can be employed to modulate NDUFC2 expression or function for therapeutic applications?

Based on the understanding that NDUFC2 deficiency contributes to disease states and that "NDUFC2 pharmacological stimulation promises to be helpful in the treatment of myocardial infarction" , several approaches can be considered for therapeutic intervention:

These therapeutic strategies require further research and validation in appropriate model systems before clinical translation, but they represent promising avenues for addressing diseases associated with NDUFC2 dysfunction, including stroke, diabetes, and certain cancers.

What are the most significant research gaps in our understanding of Macaca fascicularis NDUFC2?

Despite considerable progress in understanding NDUFC2 function and significance, several critical knowledge gaps remain:

• Detailed Structural Characterization: High-resolution structural data for NDUFC2, particularly in the context of intact Complex I from Macaca fascicularis, would provide invaluable insights into its precise interactions and functional mechanisms.

• Tissue-Specific Functions: Current research has not fully elucidated whether NDUFC2 has tissue-specific roles or interactions that might explain differential sensitivity to its deficiency across tissues.

• Regulatory Mechanisms: The mechanisms controlling NDUFC2 expression, post-translational modifications, and turnover remain poorly understood, yet are likely critical for maintaining appropriate levels of functional protein.

• Species-Specific Adaptations: While evolutionary analyses have identified selection pressure on NDUFC2, the functional consequences of species-specific amino acid changes are largely unexplored.

• Disease-Specific Mechanisms: The pathways connecting NDUFC2 deficiency to specific disease states need further clarification, particularly the mechanistic links to stroke, diabetes, and cancer.

• Therapeutic Targeting: Effective approaches for specifically modulating NDUFC2 function in vivo require development and validation.

• Population Genetics: Comprehensive analysis of NDUFC2 genetic variation across different macaque populations would enhance our understanding of its evolutionary significance.

Addressing these gaps would significantly advance both basic science understanding of mitochondrial function and the development of therapeutic interventions for NDUFC2-related disorders.

How should researchers approach the integration of NDUFC2 studies into broader mitochondrial research?

Researchers should adopt a multifaceted, integrative approach to position NDUFC2 studies within the broader context of mitochondrial research:

• Systems Biology Framework:

  • View NDUFC2 as a component within interconnected mitochondrial networks

  • Integrate NDUFC2 studies with analyses of other Complex I subunits and OXPHOS components

  • Consider mitonuclear interactions as a system rather than isolated components

• Comparative Approaches:

  • Leverage evolutionary insights from studies across primate species

  • Use the natural genetic variation in NDUFC2 across macaque populations as a tool for understanding function

  • Consider how mitonuclear interactions vary across species with different social structures

• Translational Perspective:

  • Connect findings from macaque models to human mitochondrial disorders

  • Develop biomarkers based on NDUFC2 function for disease risk assessment

  • Explore therapeutic applications of NDUFC2 modulation in relevant disease models

• Technological Integration:

  • Combine advanced techniques like complexome profiling with traditional biochemical approaches

  • Implement multi-omics strategies incorporating genomics, proteomics, and metabolomics

  • Develop in vivo imaging approaches to monitor NDUFC2 function in intact organisms

• Collaborative Research Networks:

  • Establish collaborations between evolutionary biologists, biochemists, geneticists, and clinicians

  • Share standardized protocols and reagents to enhance reproducibility

  • Develop shared databases of NDUFC2 variants and their functional characteristics