Recombinant Pongo pygmaeus NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 5, mitochondrial (NDUFB5)

What is NDUFB5 and what is its primary function in mitochondria?

NDUFB5 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). Unlike catalytic subunits, NDUFB5 is believed to serve primarily in the structural assembly and stability of Complex I rather than in direct catalysis. Complex I functions in the transfer of electrons from NADH to the respiratory chain, with ubiquinone serving as the immediate electron acceptor . The protein plays a crucial role in maintaining proper mitochondrial respiration and energy production through the electron transport chain mechanism. In Pongo pygmaeus, this protein exhibits high conservation with other mammalian homologs, reflecting the evolutionary importance of this component in mitochondrial function .

What expression systems are commonly used for producing recombinant Pongo pygmaeus NDUFB5?

Based on available research data, recombinant Pongo pygmaeus NDUFB5 is commonly expressed in yeast expression systems, which provide appropriate eukaryotic processing machinery . The choice of expression system significantly impacts protein yield, folding, and post-translational modifications. The table below compares different expression systems used for NDUFB5 production:

For research requiring high authenticity of post-translational modifications, mammalian cell expression systems may be preferred, while yeast systems offer a good balance of proper folding and cost-effectiveness .

What storage conditions are recommended for maintaining recombinant NDUFB5 stability?

Based on manufacturer recommendations, recombinant NDUFB5 should be stored at -80°C to maintain long-term stability . Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of functional activity. The protein is typically supplied in a buffer composition determined by the manufacturer, which is optimized for stability. The shelf life under appropriate storage conditions is approximately 12 months .

How can researchers effectively design experiments to study NDUFB5 interactions with other Complex I components?

Experimental design for studying NDUFB5 interactions within Complex I requires a multifaceted approach. A comprehensive experimental strategy should include:

• Co-immunoprecipitation (Co-IP): Use antibodies against NDUFB5 to pull down associated complex components, followed by mass spectrometry analysis to identify binding partners.

• Proximity labeling techniques: Employ BioID or APEX2 fusion proteins to identify proteins in close proximity to NDUFB5 within the mitochondrial membrane.

• Crosslinking mass spectrometry: Apply chemical crosslinkers to stabilize transient interactions, followed by digestion and mass spectrometry analysis to map interaction interfaces.

• Mutational analysis: Create systematic mutations in key residues to identify regions critical for protein-protein interactions within Complex I.

• Competitive binding assays: Use recombinant subunits to compete for binding with native complexes, assessing displacement effects.

For optimal experimental outcomes, researchers should consider using multiple complementary approaches rather than relying on a single method. When working specifically with Pongo pygmaeus NDUFB5, sequence differences from human NDUFB5 should be accounted for in the experimental design, particularly when using antibodies or designing fusion constructs .

What methodologies are optimal for assessing the impact of NDUFB5 on mitochondrial respiration and cell function?

To thoroughly evaluate the impact of NDUFB5 on mitochondrial respiration and cell function, researchers should employ the following methodological approaches:

• Oxygen consumption rate (OCR) measurements: Using platforms like Seahorse XF analyzers to quantify mitochondrial respiration in cells with modified NDUFB5 expression.

• Complex I activity assays: Measuring NADH:ubiquinone oxidoreductase activity using spectrophotometric assays with isolated mitochondria.

• Reactive oxygen species (ROS) quantification: Using fluorescent probes like MitoSOX to assess ROS production in response to NDUFB5 manipulation.

• Mitochondrial membrane potential assessment: Employing JC-1 or TMRM dyes to evaluate changes in mitochondrial membrane polarization.

• Cell viability and migration assays: As demonstrated in research with NDUFB5, these proteins play roles in cellular functions beyond respiration, including supporting cell viability and migration abilities .

Recent research has shown that NDUFB5 promotes cell viability, migration, and mitochondrial respiration, particularly in models of cellular stress such as Advanced Glycation End products (AGEs) exposure . This suggests a broader role for NDUFB5 in cellular resilience beyond its structural role in Complex I.

How can post-translational modifications of NDUFB5 be effectively characterized, and what is their functional significance?

Post-translational modifications (PTMs) of NDUFB5 represent an important regulatory layer that can influence protein function, stability, and interactions. Comprehensive characterization of these modifications requires:

• Mass spectrometry-based approaches:

  • High-resolution LC-MS/MS analysis after enrichment for specific modifications

  • Top-down proteomics to analyze intact protein modifications

  • Targeted multiple reaction monitoring (MRM) for quantitative analysis of specific PTMs

• Site-directed mutagenesis:

  • Systematically mutate putative modification sites to mimic or prevent modifications

  • Assess functional consequences on protein stability, complex assembly, and respiratory capacity

Recent studies have identified m6A RNA modifications as regulatory mechanisms affecting NDUFB5 expression. METTL3-mediated m6A modification has been shown to enhance NDUFB5 expression through mechanisms involving IGF2BP2 . This regulatory pathway has functional consequences, as demonstrated in the context of diabetic foot ulcer models where NDUFB5 promoted cell viability and mitochondrial function.

To assess mRNA stability influenced by these modifications, researchers can employ actinomycin D treatment to inhibit transcription, followed by quantification of NDUFB5 mRNA levels over time using qRT-PCR with primers such as:

• NDUFB5-F: 5ʹ-TCCTGTTCGACACAGTGGAG-3ʹ

• NDUFB5-R: 5ʹ-AGGACGGCCATTGTTCTTTCA-3ʹ

What are the technical considerations when using recombinant Pongo pygmaeus NDUFB5 in complex assembly studies?

When employing recombinant Pongo pygmaeus NDUFB5 in complex assembly studies, researchers should address several technical considerations:

• Tag interference assessment:

  • Evaluate whether histidine or other affinity tags affect assembly into Complex I

  • Consider using tag-removal approaches (TEV protease cleavage) if interference is detected

  • Compare assembly efficiency of tagged versus untagged proteins

• Reconstitution conditions optimization:

  • Establish optimal lipid compositions for membrane protein reconstitution

  • Determine appropriate detergent types and concentrations for solubilization

  • Optimize protein:lipid ratios for efficient complex formation

• Assembly verification methods:

  • Employ blue native PAGE to assess complex formation

  • Use analytical size exclusion chromatography to verify complex size and stability

  • Conduct functional assays to confirm the activity of assembled complexes

• Species compatibility considerations:

  • Assess compatibility of Pongo pygmaeus NDUFB5 with Complex I components from other species

  • Determine whether hybrid complexes maintain structural integrity and function

  • Identify potential interface regions that may affect cross-species assembly

What are the recommended protocols for assessing NDUFB5 expression levels in experimental systems?

For accurate quantification of NDUFB5 expression, researchers should employ a combination of complementary techniques:

• Quantitative Real-Time PCR (qRT-PCR):

  • Extract high-quality RNA using RNase-free reagents

  • Perform reverse transcription with oligo(dT) or random primers

  • Use validated NDUFB5-specific primers:

    • NDUFB5-F: 5ʹ-TCCTGTTCGACACAGTGGAG-3ʹ

    • NDUFB5-R: 5ʹ-AGGACGGCCATTGTTCTTTCA-3ʹ

  • Normalize to stable reference genes (β-actin, GAPDH)

  • Calculate relative expression using the 2^-ΔΔCT method

• Western Blotting:

  • Prepare mitochondrial fractions to enrich for target protein

  • Use specialized buffers containing appropriate detergents for membrane protein extraction

  • Employ validated antibodies specific to NDUFB5

  • Include appropriate loading controls (mitochondrial proteins like VDAC or COX IV)

• Immunocytochemistry/Immunohistochemistry:

  • Use fixation methods that preserve mitochondrial structure (e.g., 4% paraformaldehyde)

  • Employ permeabilization reagents suitable for mitochondrial proteins

  • Confirm mitochondrial localization with co-staining using established markers

When working specifically with recombinant Pongo pygmaeus NDUFB5, researchers should verify antibody cross-reactivity with the orangutan protein, as epitope differences may exist compared to human or mouse proteins that are more commonly used for antibody development .

What experimental design considerations are important when studying the role of NDUFB5 in cellular models of mitochondrial dysfunction?

When designing experiments to investigate NDUFB5's role in mitochondrial dysfunction models, researchers should consider:

• Model selection and validation:

  • Choose appropriate cell types (fibroblasts, myoblasts, neuronal cells) relevant to tissue-specific mitochondrial disorders

  • Establish baseline mitochondrial function parameters before manipulation

  • Validate models using established markers of mitochondrial dysfunction

• Intervention strategies:

  • Gene knockdown/knockout approaches (siRNA, shRNA, CRISPR-Cas9)

  • Overexpression systems (transient vs. stable expression)

  • Rescue experiments with wild-type or mutant constructs

• Technical replication and controls:

  • Include technical replicates (minimum n=3) for all experimental conditions

  • Incorporate appropriate controls (scrambled siRNA, empty vector)

  • Include positive controls (known inducers of mitochondrial dysfunction)

• Temporal considerations:

  • Determine optimal timepoints for assessing acute vs. chronic effects

  • Account for compensatory mechanisms that may emerge over time

  • Establish timeframes for protein turnover and complex assembly

• Physiologically relevant stressors:

  • Consider cellular stressors that reflect physiological challenges

  • Include models like AGEs exposure, which has been shown to affect NDUFB5-related functions

  • Incorporate metabolic challenges (glucose deprivation, galactose media)

Recent research examining NDUFB5's role in cellular responses to AGEs demonstrated that the protein promotes cell viability, migration, and mitochondrial respiration under stress conditions, suggesting experimental approaches should assess multiple cellular parameters beyond respiratory chain function .

How can recombinant NDUFB5 be utilized in the development of mitochondrial disease models and therapeutic strategies?

Recombinant NDUFB5 offers several avenues for advancing mitochondrial disease research and therapeutic development:

• Disease modeling applications:

  • Development of in vitro complementation assays for NDUFB5-related disorders

  • Creation of reporter systems to monitor Complex I assembly and function

  • Establishment of high-throughput screening platforms for compound testing

• Therapeutic strategy development:

  • Protein replacement therapy approaches using optimized delivery methods

  • Identification of small molecules that stabilize partially assembled Complex I

  • Development of gene therapy vectors for NDUFB5 expression in affected tissues

• Biomarker discovery:

  • Generation of antibodies or aptamers against specific NDUFB5 epitopes

  • Development of assays to detect pathogenic NDUFB5 variants in patient samples

  • Identification of metabolic signatures associated with NDUFB5 dysfunction

While direct NDUFB5 mutations have not been extensively documented in human mitochondrial disorders, understanding its role in Complex I assembly and function may provide insights into therapeutic approaches for related mitochondrial diseases. Comparative studies using recombinant Pongo pygmaeus NDUFB5 could help identify conserved mechanisms and therapeutic targets applicable across species .

What role does NDUFB5 play in age-related mitochondrial dysfunction and how can this be investigated experimentally?

The role of NDUFB5 in age-related mitochondrial dysfunction represents an emerging area of research that can be investigated through several experimental approaches:

• Age-dependent expression analysis:

  • Quantify NDUFB5 levels in tissues across different age groups

  • Assess post-translational modifications that may accumulate with age

  • Evaluate assembly efficiency of Complex I in young versus aged samples

• Oxidative damage assessment:

  • Examine susceptibility of NDUFB5 to oxidative modifications

  • Determine whether such modifications affect protein stability or function

  • Investigate correlation between oxidative damage to NDUFB5 and Complex I activity

• Mitochondrial quality control interactions:

  • Evaluate interactions between NDUFB5 and mitochondrial proteases or chaperones

  • Assess turnover rates in young versus aged systems

  • Determine whether NDUFB5 stability influences mitochondrial dynamics

• Comparative approaches:

  • Compare NDUFB5 characteristics between short-lived and long-lived species

  • Identify structural or functional differences that may contribute to longevity

  • Investigate whether Pongo pygmaeus NDUFB5 exhibits unique properties related to the species' relatively long lifespan

The investigation of NDUFB5 in age-related mitochondrial dysfunction may provide insights into fundamental mechanisms of aging and potential interventions to preserve mitochondrial function with advancing age .

What are the most promising future research directions for understanding NDUFB5 function and its applications?

The study of recombinant Pongo pygmaeus NDUFB5 and its homologs presents several promising research directions:

• Structural biology advancements:

  • High-resolution cryo-EM studies of Complex I with focus on NDUFB5 positioning

  • Comparative structural analysis across primate species

  • Dynamic structural studies to understand assembly processes

• Regulatory network exploration:

  • Investigation of transcriptional and post-transcriptional regulation

  • Examination of m6A modification mechanisms across species

  • Identification of factors controlling NDUFB5 expression during development and aging

• Evolutionary analysis:

  • Detailed comparative genomics across primate lineages

  • Investigation of selection pressures on NDUFB5 in different ecological niches

  • Identification of convergent evolution patterns in mitochondrial proteins

• Technological applications:

  • Development of NDUFB5-based biosensors for mitochondrial health

  • Utilization in synthetic biology approaches to enhance cellular bioenergetics

  • Application in biomimetic energy conversion systems

The exploration of NDUFB5 biology across species, particularly in non-human primates like Pongo pygmaeus, provides valuable insights into both fundamental mitochondrial biology and potential applications in biotechnology and medicine .