Recombinant Gorilla gorilla gorilla NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 (NDUFB3)

What is NDUFB3 and what is its role in mitochondrial function?

NDUFB3 (NADH:Ubiquinone Oxidoreductase Subunit B3) is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), which functions as the first enzyme in the electron transport chain of mitochondria. While classified as an accessory subunit, NDUFB3 is not directly involved in the catalytic process but is essential for proper complex assembly and stability. The protein localizes to the inner membrane of the mitochondrion as a single-pass membrane protein and contributes to the structural integrity of Complex I . Complex I facilitates the transfer of electrons from NADH to ubiquinone, which represents the initial step in the respiratory chain that ultimately leads to ATP production .

To study NDUFB3 function, researchers typically employ blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by in-gel activity assays or immunoblotting to evaluate Complex I assembly status in the presence or absence of functional NDUFB3.

How does the structure of gorilla NDUFB3 compare to human NDUFB3?

Gorilla gorilla gorilla NDUFB3 shares high sequence homology with human NDUFB3, reflecting their close evolutionary relationship. Comparative sequence analysis reveals subtle amino acid differences that may influence protein-protein interactions within Complex I. To properly investigate these differences, researchers should:

• Perform multiple sequence alignments using tools like Clustal Omega

• Generate homology models based on cryo-EM structures of mammalian Complex I

• Analyze conservation patterns at key interaction interfaces

While human NDUFB3 contains 97 amino acids, subtle species-specific variations may affect interaction with other subunits, particularly those encoded by mitochondrial DNA. These differences may reflect adaptive selection during primate evolution, as suggested by evidence of positive selection detected through both PAML and Z-test analyses .

What is known about the genomic organization of NDUFB3 across primate species?

The human NDUFB3 gene is located on chromosome 2q33.1 and consists of 4 exons spanning approximately 13.7 kb (chromosome 2; NC_000002.12 positions 201072001-201085750) . This genomic organization is generally conserved in great apes, including gorillas. When analyzing gorilla NDUFB3:

• Focus on conserved regulatory elements in the promoter region

• Examine intronic sequences for potential regulatory motifs

• Account for species-specific pseudogenes when designing primers

The human genome contains multiple NDUFB3 pseudogenes that can complicate genetic analysis . Similarly, researchers working with gorilla NDUFB3 should be aware of potential pseudogenes that may interfere with PCR amplification or sequencing. Designing primers that span exon-intron boundaries can help ensure specificity for the functional gene rather than processed pseudogenes.

What evidence suggests adaptive selection in primate NDUFB3?

Analysis of primate NDUFB3 sequences has revealed evidence of positive selection during primate evolution. Specifically, NDUFB3 showed evidence of positive selection according to Z-test analysis, though it demonstrated only borderline significance in PAML (p=0.059) . This pattern suggests that certain amino acid changes in NDUFB3 may have been adaptively selected, potentially to maintain optimal interactions with rapidly evolving mitochondrial DNA-encoded subunits of Complex I.

The adaptive selection of nuclear-encoded Complex I subunits like NDUFB3 likely reflects coevolution with mitochondrial-encoded subunits. This is consistent with the theory that nuclear genes encoding proteins that interact with mitochondrial proteins must adapt to maintain functional compatibility as mitochondrial genes evolve more rapidly .

How do researchers identify coevolutionary patterns between NDUFB3 and mtDNA-encoded Complex I subunits?

To identify coevolutionary patterns between NDUFB3 and mitochondrial DNA-encoded subunits of Complex I, researchers should:

• Perform comparative sequence analysis across multiple primate species

• Identify correlated amino acid changes between nuclear and mitochondrial subunits

• Evaluate the functional significance of co-evolving residues through biochemical assays

An example of this approach revealed coordinated changes between C39 of NDUFC2 (another nuclear-encoded Complex I subunit) and C330 of ND5 (a mitochondrial-encoded subunit) . Similar analyses could reveal important interactions between NDUFB3 and mitochondrial-encoded subunits.

Note: This table represents a conceptual framework based on the principles of coevolution between nuclear and mitochondrial genes. Specific residue interactions would require detailed structural and biochemical analysis.

How have membrane domain subunits of Complex I evolved differently from matrix domain subunits?

The membrane domain of Complex I, where NDUFB3 is located, shows distinct evolutionary patterns compared to the matrix domain. Research indicates that membrane domain subunits, including NDUFB3, have been more subject to positive selection pressure compared to matrix domain subunits . This pattern likely reflects:

• Direct interaction with rapidly evolving mtDNA-encoded subunits

• Adaptation to membrane environment variations across species

• Potential involvement in proton pumping efficiency

When studying gorilla NDUFB3, researchers should focus on residues that interact with the membrane domain and compare them with homologous positions in other primates to identify potential adaptations specific to gorilla mitochondrial function.

What expression systems are optimal for producing functional recombinant gorilla NDUFB3?

When producing recombinant gorilla NDUFB3, researchers must consider several expression systems, each with advantages and limitations:

• E. coli expression system: While commonly used for recombinant protein production, E. coli may not be optimal for NDUFB3 due to the lack of appropriate post-translational modifications and potential improper folding of membrane proteins. If using E. coli, consider fusion tags (like SUMO) to improve solubility and purification with detergents .

• Mammalian cell expression: HEK293 or CHO cells provide a more native environment for proper folding and post-translational modifications of gorilla NDUFB3, but with lower yield than bacterial systems.

• Baculovirus-insect cell system: Offers a balance between proper eukaryotic processing and reasonable yield for membrane proteins like NDUFB3.

For functional studies, mammalian or insect cell expression is recommended to ensure proper incorporation into Complex I. Researchers should include appropriate affinity tags (His-tag or FLAG-tag) for purification while confirming these do not interfere with function.

What techniques are most effective for validating recombinant NDUFB3 activity?

Validating the functionality of recombinant gorilla NDUFB3 requires multiple complementary approaches:

• Complex I assembly analysis: Blue Native PAGE followed by immunoblotting to confirm incorporation into the complete Complex I.

• Complementation assays: Introduction of recombinant gorilla NDUFB3 into NDUFB3-deficient cell lines to assess rescue of Complex I activity.

• Respiration measurements: Oxygen consumption analysis using platforms like Seahorse XF Analyzer or Oroboros Oxygraph to assess integrated mitochondrial function.

• NADH:ubiquinone oxidoreductase activity assays: Spectrophotometric measurement of Complex I enzymatic activity using NADH oxidation rate.

A comprehensive validation approach would include multiple methods, as the accessory nature of NDUFB3 means that its function is best assessed through its contribution to Complex I assembly and activity rather than through direct enzymatic assays.

How can researchers optimize the purification of recombinant gorilla NDUFB3?

Purification of recombinant gorilla NDUFB3 presents challenges due to its hydrophobic nature and membrane localization. An optimized protocol should include:

• Gentle detergent solubilization: Use mild detergents like digitonin, DDM (n-dodecyl β-D-maltoside), or LMNG (lauryl maltose neopentyl glycol) to extract NDUFB3 from membranes without denaturation.

• Two-step affinity purification: Implement sequential purification steps (e.g., His-tag followed by additional affinity tag) to enhance purity.

• Size exclusion chromatography: Further purify NDUFB3 and assess its oligomeric state.

• Stability optimization: Include cardiolipin or other lipids in buffers to maintain native-like environment.

When purifying gorilla NDUFB3, researchers should confirm protein identity by mass spectrometry and assess purity by SDS-PAGE or western blotting using antibodies against NDUFB3. For structural studies, detergent screening is crucial to identify conditions that maintain protein stability and function.

How do NDUFB3 mutations contribute to mitochondrial Complex I deficiency?

Mutations in NDUFB3 are associated with mitochondrial Complex I deficiency, nuclear type 25 . The pathogenic mechanisms include:

• Disrupted Complex I assembly: Mutations can prevent proper integration of NDUFB3 into Complex I, resulting in incomplete assembly and reduced enzyme activity.

• Structural destabilization: Certain mutations may allow assembly but compromise the structural integrity of Complex I, leading to decreased stability or altered function.

• Altered interactions with other subunits: Mutations can disrupt critical interactions with other Complex I components, particularly mitochondrial DNA-encoded subunits.

A notable example is the recurrent homozygous c.64T>C, p.Trp22Arg NDUFB3 variant identified in humans, which is associated with short stature (<9th centile) and distinctive facial features including a prominent forehead, smooth philtrum, and deep-set eyes . The clinical manifestations of NDUFB3 mutations demonstrate significant clinical variability, with some patients presenting with severe metabolic crisis while others show milder phenotypes primarily characterized by growth restriction.

How can recombinant gorilla NDUFB3 be used to model species-specific mitochondrial function?

Recombinant gorilla NDUFB3 provides an excellent tool for comparative mitochondrial research:

• Cross-species complementation studies: Introducing gorilla NDUFB3 into human or mouse cells with NDUFB3 deficiency can reveal functional differences between species.

• Chimeric Complex I analysis: Creating chimeric complexes with subunits from different species can identify specific interactions and compatibility requirements.

• Evolutionary medicine applications: Comparing the response of gorilla vs. human NDUFB3 to environmental stressors can provide insights into species-specific adaptations.

These approaches can illuminate how subtle sequence differences between gorilla and human NDUFB3 might influence mitochondrial function, potentially revealing adaptive changes that occurred during primate evolution in response to different energetic demands or environmental pressures.

What cellular phenotypes are associated with NDUFB3 dysfunction across species?

NDUFB3 dysfunction manifests in various cellular phenotypes that can be studied in experimental models:

• Reduced Complex I activity: Biochemical measurement shows decreased NADH:ubiquinone oxidoreductase activity.

• Altered mitochondrial morphology: Electron microscopy reveals changes in mitochondrial ultrastructure.

• Increased reactive oxygen species (ROS) production: Fluorescent probes demonstrate elevated oxidative stress.

• Metabolic reprogramming: Metabolomic analysis shows shifts toward glycolytic metabolism.

• Impaired mitochondrial membrane potential: Decreased membrane potential can be detected with potential-sensitive dyes.

Comparing these phenotypes between recombinant gorilla NDUFB3 models and human models can reveal species-specific responses to NDUFB3 dysfunction, potentially informing our understanding of how different primates have adapted their mitochondrial function.

How can cryo-EM approaches advance our understanding of NDUFB3's role in gorilla Complex I?

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology of large complexes and can be applied to understand species-specific aspects of NDUFB3 function:

• High-resolution structural determination: Cryo-EM can resolve the structure of gorilla Complex I with integrated NDUFB3, revealing subtle species-specific structural adaptations.

• Conformational dynamics analysis: Time-resolved cryo-EM can capture different conformational states during the catalytic cycle, showing how NDUFB3 contributes to Complex I dynamics.

• Interaction interface mapping: Structural data can precisely map interactions between NDUFB3 and other subunits, particularly those encoded by mtDNA.

These approaches require purification of intact gorilla Complex I or reconstitution with recombinant components. Comparative analysis with human Complex I structures can identify species-specific features that may reflect adaptive evolution.

What are the challenges in developing animal models for NDUFB3-related disorders?

Developing animal models to study NDUFB3 dysfunction presents several challenges:

• Embryonic lethality: Complete knockout of NDUFB3 might result in embryonic lethality due to the essential role of Complex I in energy metabolism.

• Compensatory mechanisms: Model organisms may develop compensatory mechanisms that mask the phenotypic effects of NDUFB3 mutations.

• Species-specific differences: The effects of specific mutations may differ between species due to differences in nuclear-mitochondrial interactions.

• Tissue-specific requirements: NDUFB3 dysfunction may affect tissues differently based on their energetic demands.

To address these challenges, researchers should consider conditional knockout models, tissue-specific expression systems, or introducing specific pathogenic mutations rather than complete gene deletion. Humanized mouse models expressing gorilla NDUFB3 could also provide insights into species-specific aspects of NDUFB3 function.

How might integrative multi-omics approaches enhance our understanding of NDUFB3 function?

Advanced multi-omics approaches offer powerful tools for comprehensive analysis of NDUFB3 function:

By integrating these approaches, researchers can develop a comprehensive understanding of NDUFB3's role in mitochondrial function and how species-specific variations contribute to differences in energetic metabolism between humans and gorillas.