Recombinant Mouse NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Ndufa12)

What is the molecular structure and function of mouse Ndufa12?

Mouse Ndufa12 is a core subunit of mitochondrial complex I (NADH dehydrogenase), with a molecular weight of approximately 17-20 kDa . It serves as an essential component of the electron transport chain, facilitating electron transfer from NADH to ubiquinone while contributing to the establishment of the proton gradient across the inner mitochondrial membrane . This process is fundamental to cellular energy metabolism and ATP synthesis.

The protein is encoded by the NDUFA12 gene, which produces a component of the mitochondrial respiratory chain NADH dehydrogenase (Complex I). NDUFA12 is essential for both the assembly and catalytic activity of complex I, making it crucial for mitochondrial function .

What are the recommended detection methods for mouse Ndufa12 in tissue samples?

Multiple validated detection methods are available for mouse Ndufa12, each with specific applications:

For optimal results, researchers should perform antibody titration specific to their experimental system . Polyclonal antibodies against Ndufa12 typically recognize epitopes within amino acids 200-450 of the protein and have demonstrated cross-reactivity with human, mouse, and rat samples .

How should researchers design PCR primers for mouse Ndufa12 gene expression studies?

When designing PCR primers for mouse Ndufa12 gene expression studies, researchers should consider the following methodological approach:

• Target all coding exons plus at least 10 bases of flanking noncoding DNA in all available transcripts for comprehensive coverage

• Include analysis of non-coding regions where pathogenic variants have been previously reported

• Aim for coverage depth of >20X for NGS reads or complement with Sanger sequencing

• Consider species-specific sequence variations when designing primers for cross-species studies

• Include appropriate housekeeping genes as internal controls for normalization

This approach ensures reliable quantification of Ndufa12 expression and potential identification of variants . For mutation studies, full gene coverage is essential as pathogenic variants have been identified throughout the gene sequence .

How does Ndufa12 deficiency affect mitochondrial complex I assembly and function in experimental models?

Ndufa12 deficiency significantly impairs both the assembly and function of mitochondrial complex I, with several important experimental considerations:

Biochemical analyses of Ndufa12-deficient models show isolated complex I deficiency with preserved activity of other respiratory chain complexes . Complete absence of Ndufa12 protein leads to impaired electron transfer from NADH through the respiratory chain, resulting in decreased ATP production and altered cellular energy metabolism .

Research methodologies to study these effects include:

• Blue native gel electrophoresis to assess complex I assembly status

• Enzymatic activity assays measuring NADH:ubiquinone oxidoreductase activity

• Western blot analysis to confirm Ndufa12 protein absence

• Oxygen consumption measurements to evaluate respiratory chain function

• ATP production assays to quantify energy metabolism impairment

Interestingly, despite consistent biochemical findings of Ndufa12 protein absence, clinical and phenotypic variability has been observed, suggesting the influence of additional genetic or environmental factors on disease manifestation . This makes Ndufa12 an important model for studying complex I-related diseases with variable expressivity.

What experimental approaches are most effective for investigating the role of Ndufa12 in Leigh syndrome pathophysiology?

Research into Ndufa12's role in Leigh syndrome requires a comprehensive experimental approach:

• Genetic Analysis: Next-generation sequencing combined with Sanger validation to identify and characterize novel NDUFA12 variants. Studies have revealed that homozygous mutations in NDUFA12 can lead to Leigh syndrome, with at least 6 out of 7 patients in reported families presenting with this condition .

• Protein Expression Studies: Western blot analysis has demonstrated the virtual absence of NDUFA12 protein in patient samples with homozygous mutations .

• Biochemical Characterization: Enzymatic activity assays reveal isolated complex I deficiency, which is the most frequent biochemical signature among mitochondrial diseases .

• Imaging Studies: Neuroradiological findings typically reveal bilateral symmetrical lesions in the basal ganglia and brainstem, characteristic of Leigh syndrome .

• Clinical Correlation: Despite similar biochemical profiles (absence of NDUFA12 protein), patients show variable onset and clinical progression, suggesting the influence of genetic modifiers or environmental factors .

This multi-faceted approach has expanded our understanding of genetic alterations in NDUFA12 and highlighted the phenotype variability associated with NDUFA12 defects . More than 80 monogenic causes have been implicated in Leigh syndrome, making differential diagnosis crucial in research settings .

What are the appropriate experimental controls when studying Ndufa12 function in cellular models?

Robust experimental design for Ndufa12 functional studies requires careful selection of controls:

• Positive Controls:

  • Wild-type cells/tissues known to express Ndufa12 (mouse skeletal muscle, kidney, and heart tissues have consistently shown detectable levels)

  • Recombinant Ndufa12 protein for antibody validation

  • Cells with confirmed complex I function

• Negative Controls:

  • Ndufa12 knockout models generated via CRISPR/Cas9 or siRNA knockdown

  • Isotype controls for immunostaining experiments

  • Samples from patients with confirmed NDUFA12 mutations (where available)

• Experimental Method-Specific Controls:

  • For Western blot: Loading controls (β-actin, GAPDH) and molecular weight markers

  • For immunostaining: Secondary antibody-only controls to assess non-specific binding

  • For functional assays: Pharmacological inhibitors of complex I (e.g., rotenone) as positive controls for functional impairment

• Cell Type Considerations:

  • HepG2 cells have been validated for immunofluorescence studies of Ndufa12

  • Primary cell cultures from affected tissues provide physiologically relevant models

  • iPSC-derived models offer opportunities to study tissue-specific effects

These controls ensure experimental rigor when investigating Ndufa12's role in cellular energy metabolism, complex I assembly, and mitochondrial function .

How is Ndufa12 being investigated as a potential cancer therapeutic target?

Recent research has identified Ndufa12 as a potential target for cancer therapeutics, particularly in EGFR mutant cancers. The experimental approach for investigating this target involves:

• Target Identification Techniques:

  • Drug affinity-responsive target stability (DARTS) assays combined with liquid chromatography-mass spectrometry have identified NDUFA12 as a binding protein for the natural small molecule Ertredin

  • Cellular thermal shift assays (CETSA) validated this interaction

• Functional Validation:

  • Genetic knockdown of NDUFA12 suppressed cell proliferation, mimicking the effects of Ertredin treatment

  • Studies focused particularly on EGFRvIII mutant cancer cells, where Ertredin inhibited growth and anchorage-independent 3D growth

• Mechanistic Investigations:

  • NDUFA12 appears to function as a downstream factor in EGFRvIII mutants

  • Targeting NDUFA12 may disrupt mitochondrial energy metabolism in cancer cells, representing a novel therapeutic approach

These findings suggest that NDUFA12 is a biologically relevant target for anticancer agents, offering new perspectives on mitochondrial complex I as a therapeutic target in oncology .

What methodological approaches can resolve contradictory findings in Ndufa12 phenotype-genotype correlation studies?

The reported phenotypic variability despite similar genotypic findings (homozygous NDUFA12 mutations leading to protein absence) presents a significant research challenge. To address this contradiction, researchers should consider:

• Multi-omics Integration:

  • Combine genomic, transcriptomic, proteomic, and metabolomic analyses to identify additional factors influencing disease expression

  • Search for genetic modifiers affecting complex I function or compensatory mechanisms

• Tissue-Specific Effects Investigation:

  • Compare Ndufa12 function across different tissues (brain, muscle, liver, etc.)

  • Use tissue-specific conditional knockout models to assess variable tissue sensitivities to Ndufa12 deficiency

• Environmental Factor Analysis:

  • Examine how factors like oxidative stress, metabolic state, or environmental toxins modulate phenotypic expression

  • Develop standardized protocols to control for these variables in experimental models

• Precise Phenotyping:

  • Implement comprehensive clinical assessment tools

  • Utilize advanced neuroimaging techniques for detailed characterization of Leigh syndrome progression

• Age-Dependent Effects:

  • Study developmental timing of Ndufa12 deficiency effects

  • Track longitudinal changes in mitochondrial function and clinical parameters

These methodological approaches can help elucidate the complex relationship between NDUFA12 mutations and clinical manifestations, potentially revealing therapeutic targets for intervention .

What are the recommended protocols for studying Ndufa12 interactions with other complex I components?

To study Ndufa12's interactions with other complex I components, researchers should employ these methodological approaches:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation assays using anti-Ndufa12 antibodies to pull down interacting partners

  • Proximity ligation assays to visualize in situ protein-protein interactions

  • Yeast two-hybrid or mammalian two-hybrid systems for detecting direct interactions

  • FRET/BRET analyses for real-time interaction monitoring in live cells

• Structural Biology Approaches:

  • Cryo-electron microscopy to determine the position of Ndufa12 within complex I

  • X-ray crystallography of recombinant Ndufa12 with binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Functional Interaction Assays:

  • Sequential depletion experiments (knocking down Ndufa12 followed by other complex I components)

  • Rescue experiments with mutated forms of Ndufa12 to identify critical interaction domains

  • Enzymatic assays to assess how Ndufa12 affects the function of other complex I components

• Assembly Dynamics:

  • Pulse-chase experiments to track the incorporation of Ndufa12 into complex I

  • Blue native PAGE combined with immunoblotting to identify assembly intermediates

  • Time-resolved complexome profiling to determine the sequence of assembly events

These methodological approaches will help elucidate how Ndufa12 contributes to complex I architecture, assembly, stability, and function, advancing our understanding of mitochondrial energy metabolism and potential therapeutic interventions .

What are the most promising future research avenues for Ndufa12 studies?

Based on current findings and knowledge gaps, several promising research directions for Ndufa12 studies have emerged:

• Therapeutic Development:

  • Exploration of Ndufa12 as a drug target for both mitochondrial diseases and cancer

  • Development of Ndufa12-modulating compounds based on the Ertredin model

  • Investigation of gene therapy approaches for NDUFA12-related diseases

• Complex I Assembly Mechanisms:

  • Detailed characterization of how Ndufa12 contributes to complex I assembly and stability

  • Identification of assembly factors that interact specifically with Ndufa12

  • Temporal mapping of Ndufa12 incorporation during complex I biogenesis

• Tissue-Specific Functions:

  • Investigation of why certain tissues are more affected by Ndufa12 deficiency

  • Characterization of tissue-specific Ndufa12 interactome patterns

  • Development of tissue-specific Ndufa12 conditional knockout models

• Redox Sensing and Signaling:

  • Further investigation of Ndufa12's role in oxygen sensing and hypoxic responses

  • Characterization of redox-sensitive domains within Ndufa12

  • Analysis of Ndufa12's role in cellular responses to oxidative stress

• Clinical Translation:

  • Development of biomarkers for monitoring disease progression in Ndufa12-deficient patients

  • Clinical trials of mitochondrial-targeted therapies in patients with NDUFA12 mutations

  • Implementation of newborn screening methods for early detection of NDUFA12-related disorders

These research directions will contribute to a more comprehensive understanding of Ndufa12 function and potentially lead to novel therapeutic strategies for mitochondrial diseases and cancer .

What methodological challenges must be addressed to advance Ndufa12 research?

Advancing Ndufa12 research requires addressing several key methodological challenges:

• Model System Limitations:

  • Development of physiologically relevant models that accurately recapitulate human NDUFA12-related diseases

  • Creation of conditional and inducible knockout systems to study acute versus chronic effects of Ndufa12 deficiency

  • Generation of patient-derived iPSCs harboring NDUFA12 mutations for disease modeling

• Technical Hurdles:

  • Improving antibody specificity for detecting low Ndufa12 expression levels

  • Developing methods to study the dynamic assembly of complex I in living cells

  • Creating assays to measure tissue-specific impacts of Ndufa12 deficiency

• Standardization Issues:

  • Establishing standardized protocols for measuring complex I activity

  • Developing consensus guidelines for phenotyping Ndufa12-deficient models

  • Creating reference datasets for interpreting complex I assembly patterns

• Translational Barriers:

  • Bridging the gap between basic Ndufa12 research and clinical applications

  • Developing targeted delivery systems for complex I-directed therapeutics

  • Implementing personalized medicine approaches for patients with NDUFA12 mutations

• Integrated Analysis Challenges:

  • Developing computational tools to integrate multi-omics data from Ndufa12 studies

  • Creating systems biology models of how Ndufa12 deficiency affects cellular metabolism

  • Implementing machine learning approaches to predict phenotypic outcomes based on specific NDUFA12 variants