Recombinant Chicken NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 4 (NDUFB4)

What is the basic structure of chicken NDUFB4 and how does it compare to mammalian homologs?

Chicken NDUFB4 is a hydrophobic subunit of mitochondrial Complex I (NADH dehydrogenase). Similar to its mammalian counterparts, it has a characteristic two-domain structure consisting of an N-terminal hydrophobic domain that forms an alpha helix spanning the inner mitochondrial membrane, and a C-terminal hydrophilic domain that interacts with globular subunits of Complex I . The highly conserved nature of this structure across species suggests that chicken NDUFB4 shares significant structural homology with human NDUFB4, which is a 15 kDa protein composed of 129 amino acids . When studying chicken NDUFB4, researchers should be aware that while the core structure is likely conserved, species-specific differences may affect antibody binding sites and interaction partners.

How is NDUFB4 expression regulated in chicken tissues?

The regulation of NDUFB4 expression in chicken tissues appears to be tissue-specific and can be altered during viral infections such as Marek's Disease Virus (MDV) . While detailed chicken-specific expression patterns are still being characterized, studies in mammals suggest that NDUFB4 expression is coordinated with other mitochondrial Complex I subunits to maintain proper stoichiometry. In chickens, NDUFB4 expression changes have been observed in response to MDV infection, although the regulatory mechanisms remain to be fully elucidated . When designing expression studies, researchers should consider tissue-specific differences and potential viral-induced changes in expression patterns.

What are the optimal methods for recombinant expression of chicken NDUFB4?

• Codon optimization: Adapt codons for the expression host to improve translation efficiency

• Fusion tags: Include a C-terminal His6 or FLAG tag for purification, avoiding N-terminal tags that might interfere with membrane insertion

• Induction conditions: For E. coli systems, use lower IPTG concentrations (0.1-0.5 mM) and temperatures (16-20°C) to reduce inclusion body formation

• Membrane extraction: Employ gentle detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin for solubilization

Similar approaches have been successful for expression of NDUFB4 in other systems, as demonstrated in studies of NDUFB4 mutants .

What purification strategies yield the highest purity and activity for recombinant chicken NDUFB4?

Purification of chicken NDUFB4 requires specialized approaches due to its hydrophobic nature and membrane association. A multi-step purification protocol is recommended:

• Initial solubilization: Use digitonin (1-2%) or DDM (0.5-1%) to extract from membranes without denaturing the protein

• Affinity chromatography: Utilize His-tag or FLAG-tag affinity as the first purification step

• Size exclusion chromatography: Separate monomeric NDUFB4 from aggregates or other complexes

• Ion exchange chromatography: As a final polishing step if higher purity is required

To maintain functional activity, all buffers should contain 0.05-0.1% digitonin or DDM and optimization of salt concentration (typically 150-300 mM NaCl) is essential. Researchers should validate purification efficiency using techniques such as BN-PAGE, which has been effectively used to analyze NDUFB4 in complex assemblies .

How can researchers effectively validate the functional integrity of purified chicken NDUFB4?

Functional validation of purified chicken NDUFB4 should assess both its structural integrity and ability to participate in Complex I assembly. A comprehensive validation approach includes:

• Complex I incorporation assay: Using BN-PAGE and immunoblotting to verify incorporation into Complex I when added to NDUFB4-depleted mitochondrial membranes

• Supercomplex formation analysis: Assessing the ability to promote supercomplex formation, particularly I₁III₂IV₁ respirasome assembly

• Respiration measurements: Using oxygen consumption assays to measure functional restoration of electron transport in NDUFB4-deficient mitochondria

• Biophysical characterization: Circular dichroism to confirm alpha-helical content consistent with proper folding

• Interaction validation: Pull-down assays to verify binding to known interaction partners such as Complex III subunit UQCRC1

Researchers should compare their purified protein to positive controls and establish clear acceptance criteria for functional integrity before proceeding with downstream applications.

How does NDUFB4 expression change during Marek's Disease Virus infection in chickens?

During Marek's Disease Virus (MDV) infection in chickens, mitochondrial function undergoes significant alterations that may involve changes in NDUFB4 expression. While the specific regulation of NDUFB4 during MDV infection is not fully characterized, studies have shown that MDV infection affects the expression of numerous mitochondria-related genes in chickens . The response appears to be chicken line-dependent, with different genetic backgrounds showing varying patterns of mitochondrial gene expression in response to infection .

In line 7₂ chickens, which are more susceptible to MDV-induced tumor formation, significant changes in mitochondria-related nuclear genes were observed post-infection, with most genes being upregulated, particularly in the spleen . While NDUFB4 was mentioned among the examined mitochondrial genes, its specific expression pattern requires further investigation. Researchers studying viral effects on mitochondrial function should consider examining NDUFB4 regulation as part of a broader assessment of mitochondrial alterations.

What role might NDUFB4 play in avian metabolic disorders compared to mammalian models?

Based on mammalian studies, NDUFB4 plays an important role in maintaining respiratory supercomplex integrity, particularly the I₁III₂IV₁ respirasome . Alterations in this protein might therefore contribute to metabolic disorders in avian species, albeit with potential differences from mammalian models. Unlike NDUFS4, which shows consistent downregulation in diabetic kidney disease in mammals , NDUFB4 was not consistently downregulated in diabetic models . This suggests that different Complex I subunits may have distinct roles in metabolic pathology.

In avian models, NDUFB4 dysfunction might manifest differently due to birds' unique metabolic adaptations for flight and higher body temperatures. Researchers investigating avian metabolic disorders should consider:

• Higher baseline metabolic rates in birds may make them more sensitive to NDUFB4 dysfunction

• Avian-specific energy demands might lead to tissue-specific effects not observed in mammals

• NDUFB4 interactions with avian-specific proteins could create unique pathophysiological mechanisms

Comparative studies between mammalian and avian NDUFB4 function could provide valuable insights into both conserved and species-specific roles in metabolic regulation.

How can researchers effectively use NDUFB4 genetic modifications to study mitochondrial supercomplex formation in avian models?

Studying mitochondrial supercomplex formation in avian models through NDUFB4 genetic modifications requires sophisticated approaches that preserve Complex I assembly while specifically targeting supercomplex formation. Based on successful mammalian studies , researchers can employ these strategies:

• Point mutation approach: Target conserved residues like N24 and R30 that interact with Complex III components rather than those essential for Complex I assembly

• Conditional expression systems: Use tetracycline-inducible or similar systems to control timing and degree of mutation expression

• Domain-specific modifications: Engineer chimeric constructs replacing only the interaction domains between NDUFB4 and Complex III components

• Tissue-specific targeting: Use avian-appropriate promoters for tissue-restricted expression to avoid systemic effects

The effectiveness of these modifications can be monitored through:

These approaches enable researchers to specifically attribute phenotypes to supercomplex disruption rather than general Complex I dysfunction .

What are the most effective protocols for investigating NDUFB4's role in mitochondrial cristae remodeling in chicken cells?

Investigating NDUFB4's role in mitochondrial cristae remodeling in chicken cells requires integrating advanced imaging techniques with functional and molecular analyses. While NDUFB4-specific effects on cristae have not been directly established, approaches similar to those used in studies of NDUFS4's effects on cristae remodeling would be applicable:

• High-resolution imaging protocol:

  • Transmission electron microscopy of mitochondria from NDUFB4-modified cells

  • Quantification of cristae density, width, and junction diameter

  • Correlative light and electron microscopy to link NDUFB4 localization with cristae morphology

• Protein interaction analysis:

  • Co-immunoprecipitation of NDUFB4 with known cristae-shaping proteins (OPA1, Mic60)

  • Proximity labeling (BioID or APEX) to identify chicken-specific NDUFB4 interaction partners

• Dynamic assessment:

  • Live-cell super-resolution imaging of mitochondria using appropriate fluorescent markers

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to assess NDUFB4 mobility

• Functional correlates:

  • Membrane potential measurements using potentiometric dyes

  • Local ATP production assessment using genetically-encoded ATP sensors

These methods should be applied to both normal and stressed conditions (e.g., high glucose, hypoxia) to reveal context-dependent roles of NDUFB4 in cristae remodeling, similar to the approach used in diabetic models with NDUFS4 .

How does chicken NDUFB4 interact with the mtDNA replication and transcription machinery under metabolic stress?

The interaction between chicken NDUFB4 and mtDNA machinery under metabolic stress represents an advanced research area that connects respiratory chain function with mitochondrial genome maintenance. While direct evidence for NDUFB4-specific effects is limited, research approaches should consider:

• Proximity-based interaction mapping:

  • BioID or APEX2 fusion proteins to identify stress-dependent interactions

  • Cross-linking mass spectrometry to capture transient associations with mtDNA replication/transcription factors

• mtDNA dynamics assessment:

  • Quantification of mtDNA copy number changes in NDUFB4-deficient cells under metabolic challenges

  • Analysis of mtDNA replication intermediates using 2D-AGE (two-dimensional agarose gel electrophoresis)

• Transcriptional impact analysis:

  • RNA-seq to profile expression changes in mtDNA-encoded genes

  • Measurement of mtDNA-encoded transcript stability and processing

In chicken models, metabolic stress conditions of particular relevance would include:

Research in this area should specifically examine whether NDUFB4 dysfunction affects expression of key mtDNA maintenance factors such as TFAM, POLG, TWNK, and SSBP1, which have been shown to be dysregulated in response to mitochondrial stress in other models .

How has NDUFB4 evolved across avian lineages compared to mammalian evolution?

Evolutionary analysis of NDUFB4 across avian lineages reveals important insights into mitochondrial adaptation. While specific chicken NDUFB4 evolutionary data is limited, comparative approaches should explore:

• Sequence conservation patterns:

  • Higher conservation of membrane-spanning domains compared to matrix-exposed regions

  • Conservation of key interaction residues (e.g., N24, R30) involved in supercomplex formation

  • Lineage-specific variations that correlate with metabolic adaptations (e.g., migratory vs. non-migratory birds)

• Selection pressure analysis:

  • Calculation of dN/dS ratios across bird orders with different metabolic demands

  • Identification of positively selected sites that might reflect adaptation to flight or thermal regulation

• Structure-function relationships:

  • Homology modeling of NDUFB4 from diverse avian species

  • Correlation of structural variations with species-specific metabolic parameters

Compared to mammalian evolution, avian NDUFB4 likely shows adaptations reflecting the high-energy demands of flight and higher body temperatures. Researchers examining evolutionary aspects should consider that the functional constraints on NDUFB4 may differ between avian and mammalian lineages due to these metabolic differences.

What methodological approaches are most effective for comparing NDUFB4 function between chicken and mammalian systems?

Effective comparison of NDUFB4 function between chicken and mammalian systems requires complementary approaches that account for species-specific differences while maintaining comparable experimental conditions. Recommended methodological approaches include:

• Heterologous expression systems:

  • Expression of chicken NDUFB4 in mammalian NDUFB4-knockout cells to assess functional complementation

  • Reciprocal expression of mammalian NDUFB4 in chicken cells

  • Creation of chimeric proteins with domain swapping between species

• Equivalent measurement conditions:

  • Standardization of temperature parameters (chicken mitochondria typically function at higher temperatures)

  • Adjustment of substrate concentrations to account for species-specific metabolic rates

  • Consistent isolation methods for mitochondria from both systems

• Comparative structural analysis:

  • Cryo-EM of respiratory complexes from both species under identical conditions

  • Cross-linking mass spectrometry to identify conserved and divergent interaction partners

• Functional assessments:

  • High-resolution respirometry with identical substrate combinations

  • Measurement of ROS production under comparable stress conditions

  • Assessment of supercomplex formation efficiency using BN-PAGE with standardized solubilization conditions

These approaches enable valid cross-species comparisons while accounting for the biological differences between avian and mammalian systems.

What are the major technical challenges in studying chicken NDUFB4 and how can they be overcome?

Studying chicken NDUFB4 presents several technical challenges that require specialized approaches. Major challenges and their solutions include:

• Antibody availability and specificity:

  • Challenge: Limited commercial antibodies for chicken NDUFB4

  • Solution: Generate custom antibodies against conserved epitopes or use epitope tags in recombinant systems; validate antibody specificity using NDUFB4-knockout controls

• Membrane protein solubilization:

  • Challenge: NDUFB4's hydrophobicity complicates extraction and analysis

  • Solution: Optimize detergent conditions (digitonin 1-2% or DDM 0.5-1%) for gentle solubilization; use styrene-maleic acid lipid particles (SMALPs) for detergent-free extraction

• Maintaining respiratory complex integrity:

  • Challenge: Complex I easily dissociates during isolation

  • Solution: Use mild solubilization conditions and stabilizing agents; perform analyses at physiologically relevant temperatures for chicken (40-42°C)

• Genetic manipulation of chicken cells:

  • Challenge: Lower efficiency of genetic tools in avian cells

  • Solution: Optimize CRISPR/Cas9 systems for chicken cells; use chicken-derived cell lines (e.g., DF-1) for higher transfection efficiency

• Functional assessment in avian systems:

  • Challenge: Standard respirometry protocols may not be optimized for avian mitochondria

  • Solution: Adjust temperature and substrate conditions to match avian physiology; develop avian-specific normal ranges for mitochondrial parameters

These technical solutions enable more effective study of chicken NDUFB4 while maintaining experimental rigor comparable to mammalian systems.

How can researchers troubleshoot expression and activity issues with recombinant chicken NDUFB4?

Troubleshooting expression and activity issues with recombinant chicken NDUFB4 requires systematic analysis of the expression system, purification process, and functional assays. This table outlines common problems and their solutions:

When addressing these issues, researchers should implement changes systematically and validate improvements at each step using appropriate analytical techniques such as BN-PAGE, Western blotting, and functional assays .

What emerging technologies will advance our understanding of chicken NDUFB4 function in mitochondrial biology?

Several cutting-edge technologies are poised to transform our understanding of chicken NDUFB4 function in mitochondrial biology:

• Cryo-electron tomography for visualizing NDUFB4 in its native membrane environment, revealing species-specific supercomplex arrangements at sub-nanometer resolution

• AlphaFold2 and related AI-based structural prediction tools for modeling chicken-specific NDUFB4 structures and interactions, particularly useful for regions divergent from mammalian homologs

• Single-molecule FRET for examining dynamic interactions between NDUFB4 and other respiratory complex components under various metabolic conditions

• Genome-wide CRISPR screens in chicken cell lines to identify synthetic lethal interactions with NDUFB4 dysfunction, revealing compensatory pathways

• Spatially-resolved transcriptomics and proteomics for mapping tissue-specific expression patterns and interactomes of NDUFB4 in developing and adult chickens

• Organoid models derived from chicken stem cells to study NDUFB4 function in tissue-specific contexts under controlled conditions

• In situ structural techniques like proximity labeling combined with mass spectrometry for mapping the protein neighborhood of NDUFB4 in intact mitochondria

These emerging technologies will enable researchers to move beyond traditional biochemical approaches and develop a more integrated understanding of NDUFB4's role in chicken mitochondrial biology, with potential applications to both basic science and poultry health.

What are the critical unanswered questions regarding chicken NDUFB4's role in mitochondrial disease models?

Several critical questions remain unanswered regarding chicken NDUFB4's role in mitochondrial disease models that will shape future research:

• Line-specific susceptibility: How do genetic differences in NDUFB4 between chicken lines affect susceptibility to mitochondrial dysfunction during viral infections like MDV ?

• Tissue-specific effects: Why does NDUFB4 dysfunction manifest differently across tissues, particularly in response to stressors like viral infection ?

• Supercomplex regulation: How does chicken NDUFB4 contribute to dynamic supercomplex assembly and disassembly under changing metabolic conditions ?

• Metabolic adaptation: Does NDUFB4 play a role in the unique metabolic adaptations of avian species, such as their higher body temperature and metabolic rate?

• Therapeutic potential: Could targeted modulation of NDUFB4 function protect against mitochondrial dysfunction in avian disease models?

• Developmental regulation: How is NDUFB4 expression regulated during embryonic development, and does it contribute to tissue-specific mitochondrial maturation?

• Environmental response: How do environmental stressors relevant to poultry production (heat, crowding, nutrition) impact NDUFB4 function and mitochondrial health?

Addressing these questions will require integrated approaches combining molecular, cellular, and in vivo methodologies, with particular attention to avian-specific aspects of mitochondrial biology that may not be captured in mammalian models.

How can recombinant chicken NDUFB4 be utilized as a tool to study respiratory supercomplex assembly?

Recombinant chicken NDUFB4 offers unique opportunities for studying respiratory supercomplex assembly through several experimental approaches:

• In vitro reconstitution assays:

  • Purified recombinant NDUFB4 can be added to NDUFB4-depleted mitochondrial membranes to study its role in facilitating supercomplex formation

  • The effect of site-directed mutations (particularly at residues homologous to human N24 and R30) on supercomplex assembly can be systematically assessed

• Competition experiments:

  • Wild-type and mutant versions of recombinant NDUFB4 can be used in competition assays to determine binding affinities and assembly preferences

  • Tagged versions can track the kinetics of incorporation into existing complexes

• Cross-linking studies:

  • Recombinant NDUFB4 with incorporated photo-activatable amino acids at specific positions can map interaction interfaces with Complex III components

  • Chemical cross-linking followed by mass spectrometry can identify species-specific interaction partners

• Structural biology applications:

  • Purified recombinant NDUFB4 can be used for crystallization trials or cryo-EM studies in combination with other complex components

  • Structural information can inform the design of interface-specific inhibitors or stabilizers

These approaches provide valuable tools for understanding the species-specific aspects of supercomplex assembly that may be particularly relevant to avian mitochondrial function and bioenergetics .

What experimental design considerations are essential when studying chicken NDUFB4 in the context of viral infections like MDV?

When studying chicken NDUFB4 in the context of viral infections like Marek's Disease Virus (MDV), several critical experimental design considerations must be addressed:

• Chicken line selection:

  • Include both resistant (line 6₃) and susceptible (line 7₂) chicken lines, as they show distinct mitochondrial responses to MDV infection

  • Document genetic background thoroughly, as mitochondrial responses vary significantly between lines

• Tissue sampling strategy:

  • Examine multiple tissues (spleen, thymus, bursa) as NDUFB4 responses may be tissue-specific

  • Consider time-course sampling to capture dynamic changes in expression and function

• Appropriate controls:

  • Include age-matched, uninfected controls from the same genetic background

  • Consider sham-inoculated controls to account for injection stress

• Comprehensive analysis approach:

  • Integrate transcriptomic, proteomic, and functional assessments of mitochondria

  • Measure both NDUFB4 expression and its incorporation into Complex I and supercomplexes

• Viral strain and dose standardization:

  • Document MDV strain, passage history, and dose for reproducibility

  • Consider comparing responses to vaccine strains vs. virulent strains

• Mitochondrial functional assessment:

  • Measure respiratory function, supercomplex assembly, and ROS production

  • Assess mitochondrial morphology and distribution in infected cells

This multifaceted experimental design enables researchers to distinguish direct viral effects on NDUFB4 from secondary metabolic adaptations, providing insight into how mitochondrial function influences viral pathogenesis in avian hosts .

How do findings from NDUFB4 studies inform our broader understanding of avian mitochondrial biology?

Research on chicken NDUFB4 provides important insights that expand our understanding of avian mitochondrial biology in several key dimensions:

• Supercomplex organization: Studies demonstrating NDUFB4's role in respirasome formation suggest that the spatial organization of respiratory complexes is evolutionarily conserved but may have avian-specific features that accommodate their higher metabolic demands.

• Stress response mechanisms: The differential regulation of mitochondrial genes, including potential changes in NDUFB4, during MDV infection reveals how avian mitochondria respond to pathological stress, potentially informing broader concepts of mitochondrial adaptation.

• Species-specific bioenergetics: Comparative analyses of NDUFB4 function between avian and mammalian systems highlight adaptations that may support birds' higher body temperatures and metabolic rates.

• Tissue-specific regulation: The observation that mitochondrial responses to MDV differ between tissues suggests tissue-specific regulation of mitochondrial function that may be mediated partly through accessory subunits like NDUFB4.

• Genetic background effects: The dramatic differences in mitochondrial responses between chicken lines with different susceptibility to MDV demonstrate how genetic background influences mitochondrial function, with potential implications for breeding and genetic selection.

These findings collectively enhance our understanding of mitochondrial biology beyond individual subunits, revealing principles of metabolic regulation and adaptation that are relevant to both basic science and applied poultry research.

What are the implications of chicken NDUFB4 research for applied poultry science and veterinary medicine?

Research on chicken NDUFB4 has several important implications for applied poultry science and veterinary medicine:

• Biomarker development: Changes in NDUFB4 expression or function could serve as early biomarkers for mitochondrial dysfunction in production settings, potentially allowing early intervention before clinical disease manifestation.

• Disease resistance breeding: Understanding how NDUFB4 and related mitochondrial genes differ between disease-resistant and susceptible chicken lines could inform genetic selection strategies to enhance natural resistance to pathogens like MDV.

• Nutritional interventions: Knowledge of how NDUFB4 contributes to mitochondrial efficiency could guide the development of feed supplements that support optimal mitochondrial function during growth, production stress, or disease challenges.

• Vaccine development: Insights into how viral infections like MDV affect mitochondrial function through changes in proteins like NDUFB4 may reveal new targets for attenuated vaccine development or adjuvant strategies.

• Environmental stress management: Understanding the relationship between NDUFB4 function and mitochondrial responses to stressors could inform housing and management practices that minimize physiological stress in production environments.

• Therapeutic approaches: In the longer term, strategies to modulate specific aspects of mitochondrial function through targeted regulation of components like NDUFB4 might provide new therapeutic approaches for mitochondrial dysfunction in poultry.