Recombinant Gorilla gorilla gorilla NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3 (NDUFA3)

What is NDUFA3 and what is its fundamental role in mitochondrial function?

NDUFA3, also known as Complex I-B9 or NADH-ubiquinone oxidoreductase B9 subunit, is an essential subunit of mitochondrial electron transport chain Complex I. It plays a crucial role in facilitating oxidative phosphorylation, the process fundamental to ATP production in aerobic organisms . As part of the NADH dehydrogenase complex, NDUFA3 participates in electron transfer from NADH to ubiquinone, contributing to the proton gradient generation across the inner mitochondrial membrane that ultimately drives ATP synthesis.

The protein's evolutionary conservation across primates, including humans, chimpanzees, and gorillas, underscores its critical importance in cellular energy metabolism . Functionally, NDUFA3 contributes to maintaining proper electron flow through Complex I, which when disrupted can lead to increased reactive oxygen species (ROS) production and subsequent oxidative damage, as demonstrated in high glucose-induced cellular injury models .

What is the molecular structure and composition of Gorilla gorilla gorilla NDUFA3?

Gorilla gorilla gorilla NDUFA3 is a full-length protein comprising 84 amino acids with the following sequence: MAARVGAFLKNAWDKEPVLVVSFVVGGLAVILPPLSPYFKYSVMINKATPYNYPVPVRDDGNMPDVPSHPQDPQGPSLEWLKKL . The recombinant form can be produced with an N-terminal 10xHis-tag to facilitate purification and experimental manipulation.

Despite its relatively small size, NDUFA3 serves a significant structural role in Complex I assembly and stability. Its hydrophobic amino acid content suggests membrane-associated domains that facilitate integration into the Complex I substructure. For research applications, the protein is typically available in either liquid form or as a lyophilized powder, with the latter offering greater stability and a longer shelf life (approximately 12 months at -20°C/-80°C compared to 6 months for liquid formulations) .

How does NDUFA3 differ between gorillas, humans, and other primates from an evolutionary perspective?

Comparative studies examining NDUFA3 across primate species have revealed interesting evolutionary patterns. Research analyzing mitochondrial DNA variation in humans, chimpanzees, and gorillas demonstrated that within humans and within chimpanzees, the ratio of replacement to silent nucleotide substitutions is higher than observed in comparisons between species . This pattern, observed across the mitochondrial genome including genes like NDUFA3, contradicts neutral evolution expectations.

This finding suggests that many mitochondrial protein polymorphisms are slightly deleterious, aligning with observations from human mitochondrial disease studies . Such evolutionary patterns indicate that while NDUFA3 is functionally conserved due to its essential role in energy metabolism, species-specific variations likely reflect adaptations to different metabolic requirements or environmental pressures. These comparative studies provide valuable insights for researchers investigating the functional consequences of NDUFA3 variations in different primate models.

What are the recommended methods for expressing and purifying recombinant NDUFA3?

For successful expression of recombinant Gorilla gorilla gorilla NDUFA3, an in vitro Escherichia coli expression system has proven effective . The recommended approach involves:

• Expression construct design: Incorporation of the full coding sequence (amino acids 1-84) with an N-terminal 10xHis-tag to facilitate purification

• Expression conditions: Optimization of temperature, induction time, and media composition for maximum protein yield

• Purification protocol: Sequential affinity chromatography using the His-tag, followed by size exclusion chromatography to ensure protein homogeneity

• Buffer composition: Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has been demonstrated to maintain protein stability

For functional studies, it's essential to verify that the recombinant protein retains native conformation through circular dichroism spectroscopy or limited proteolysis. Activity validation can be performed through assays measuring electron transfer capacity or Complex I assembly efficiency. Researchers should prepare working aliquots to minimize freeze-thaw cycles, as repeated freezing and thawing significantly reduces protein stability and activity .

How can researchers design knockout or knockdown experiments to study NDUFA3 function?

Designing effective knockout or knockdown experiments for NDUFA3 requires careful consideration of both technical approach and biological consequences:

• CRISPR-Cas9 knockout: Successfully employed to generate NDUFA3 knockout cell lines in HEK293 cells . Guide RNA design should target conserved regions, with validation of knockout efficiency at both genomic (sequencing) and protein (Western blot) levels.

• RNA interference approaches: siRNA or shRNA targeting NDUFA3 provides an alternative for transient or stable knockdown, particularly valuable when complete knockout might compromise cell viability.

• Inducible systems: Given NDUFA3's critical role in mitochondrial function, inducible knockout/knockdown systems (e.g., Tet-On/Off) allow temporal control to circumvent potential lethality.

• Functional assessment: Comprehensive evaluation should include multiple parameters:

  • Mitochondrial complex I activity assays

  • Oxygen consumption rate (OCR) measurements

  • Reactive oxygen species (ROS) quantification

  • Mitochondrial membrane potential assessment

  • Cell viability and apoptosis markers

For phenotypic interpretation, researchers should consider potential compensatory mechanisms that might emerge following NDUFA3 depletion, particularly upregulation of other Complex I subunits or alternative metabolic pathways.

What are critical parameters to monitor when studying NDUFA3 in the context of mitochondrial dysfunction models?

When examining NDUFA3 in mitochondrial dysfunction models, researchers should monitor multiple parameters to comprehensively assess functional consequences:

• Bioenergetic profile: Oxygen consumption rate (OCR) provides critical information about mitochondrial respiration capacity. High glucose-induced mitochondrial dysfunction models show decreased OCR values, which can be counteracted by NDUFA3 overexpression .

• Oxidative stress markers: NDUFA3 manipulation directly impacts ROS production. Both intracellular ROS levels and markers of oxidative damage (protein carbonylation, lipid peroxidation, DNA oxidation) should be quantified.

• Mitochondrial membrane potential (MMP): As demonstrated in high glucose-treated cells, conditions compromising NDUFA3 function can reduce MMP , affecting numerous mitochondrial processes.

• Complex I activity: Direct measurement of NADH:ubiquinone oxidoreductase activity provides specific assessment of the complex containing NDUFA3.

• Cell death parameters: NDUFA3 dysfunction can trigger apoptotic pathways, necessitating monitoring of cell viability, apoptotic markers (Annexin V, caspase activation), and necroptic indicators.

• Adaptive responses: Changes in mitochondrial biogenesis (PGC-1α, TFAM expression), mitophagy markers, and compensatory metabolic pathways should be assessed to understand cellular adaptation to NDUFA3 perturbation.

Including appropriate controls is essential, particularly treatment with specific mitochondrial inhibitors like rotenone (Complex I inhibitor) to provide benchmarks for comparison with NDUFA3-mediated effects .

How does NDUFA3 contribute to cellular responses to oxidative stress and mitochondrial dysfunction?

NDUFA3 plays a significant role in cellular responses to oxidative stress through its function in mitochondrial Complex I. Research demonstrates that modulation of NDUFA3 expression directly impacts reactive oxygen species (ROS) production and mitochondrial function under stress conditions. In human nucleus pulposus cells exposed to high glucose, NDUFA3 overexpression effectively counteracted increased ROS production, decreased mitochondrial membrane potential, and reduced oxygen consumption rate .

The protective mechanism likely involves:

• Maintenance of efficient electron transfer through Complex I, preventing electron leakage that would otherwise generate superoxide radicals

• Preservation of mitochondrial membrane potential, essential for proper mitochondrial function and prevention of apoptotic cascades

• Support of oxygen consumption rate, maintaining efficient ATP production under metabolic stress conditions

These findings suggest NDUFA3 as a potential therapeutic target in conditions characterized by mitochondrial dysfunction and oxidative stress. Researchers investigating NDUFA3's role should employ comprehensive approaches measuring multiple oxidative stress parameters alongside mitochondrial function assessments to fully characterize its protective effects .

What is the relationship between NDUFA3 and metabolic disorders such as diabetes mellitus?

Evidence supports a significant relationship between NDUFA3 and metabolic disorders, particularly diabetes mellitus. Research has demonstrated that diabetes mellitus represents a well-documented risk factor for intervertebral disc degeneration, with mitochondrial dysfunction as an underlying mechanism . In this context, NDUFA3 plays a protective role against high glucose-induced cellular injuries.

Key findings regarding this relationship include:

• High glucose conditions decrease cell viability, increase apoptotic cell death, elevate ROS production, and compromise mitochondrial function in a dose-dependent manner

• NDUFA3 overexpression effectively counteracts these high glucose-induced injuries, preserving mitochondrial function and reducing oxidative stress

• The protective effects of NDUFA3 are mediated through maintaining mitochondrial Complex I activity, which is compromised in diabetic conditions

• HDAC/H3K27ac mechanisms are involved in regulating NDUFA3 transcription, suggesting epigenetic regulatory pathways that could be targeted therapeutically

These findings position NDUFA3 as a potential therapeutic target for diabetes-related complications, particularly those involving mitochondrial dysfunction. Future research directions should include investigation of NDUFA3 expression patterns in diabetic tissues and potential pharmacological approaches to enhance NDUFA3 function in metabolic disorders.

How can NDUFA3 genetic variations be studied in the context of evolutionary mitochondrial genomics?

Studying NDUFA3 genetic variations in evolutionary mitochondrial genomics requires a multifaceted approach combining comparative genomics, population genetics, and functional analysis:

• Comparative sequence analysis: Existing research has sequenced NDUFA3 from humans, chimpanzees, and gorillas, revealing distinct patterns of nucleotide substitution . Expanding these analyses to additional primate species would provide greater evolutionary context.

• Substitution pattern analysis: The ratio of replacement to silent nucleotide substitutions within and between species offers insights into selection pressures. The higher ratio observed within species compared to between species for mitochondrial genes like NDUFA3 contradicts neutral evolution models .

• Integration with mitochondrial haplotype data: Analyzing NDUFA3 variations within the context of broader mitochondrial haplotypes can reveal co-evolutionary patterns among respiratory complex components.

• Functional consequences assessment: Recombinant expression of NDUFA3 variants identified across species, followed by functional characterization of Complex I activity, electron transfer efficiency, and ROS production provides insights into adaptive significance.

• Population-specific variation analysis: Examining human population-specific NDUFA3 variants can identify potential adaptations to different environmental conditions or metabolic demands.

This research approach could reveal how evolutionary pressures have shaped mitochondrial function across primates and identify potentially adaptive variations in NDUFA3 that could inform therapeutic strategies for mitochondrial disorders.

What are the optimal conditions for storing and handling recombinant NDUFA3 protein preparations?

Optimal storage and handling of recombinant NDUFA3 require careful attention to multiple factors to preserve protein stability and functionality:

• Storage temperature: Maintain at -20°C/-80°C for maximum stability. Lyophilized formulations can be stored for approximately 12 months, while liquid formulations typically remain stable for about 6 months under these conditions .

• Buffer composition: Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has demonstrated effectiveness in maintaining NDUFA3 stability . The trehalose component serves as a cryoprotectant that prevents protein denaturation during freeze-thaw cycles.

• Aliquoting strategy: Prepare small working aliquots immediately upon receiving or reconstituting the protein to minimize freeze-thaw cycles, which significantly compromise protein integrity.

• Thawing protocol: Rapidly thaw frozen aliquots at room temperature rather than slow thawing at 4°C to minimize protein aggregation.

• Handling precautions:

  • Avoid vigorous vortexing that can denature the protein

  • Maintain sterile technique to prevent microbial contamination

  • Use low-binding microcentrifuge tubes to minimize protein adherence to tube walls

  • Centrifuge briefly after thawing to collect any condensation

• Quality control: Before experimental use, verify protein integrity through SDS-PAGE and functional activity assays, particularly for samples stored for extended periods .

Adherence to these guidelines ensures maximum retention of NDUFA3 structural integrity and functional activity in research applications.

What techniques are most effective for analyzing NDUFA3 interactions with other mitochondrial proteins?

Elucidating NDUFA3 interactions with other mitochondrial proteins requires complementary approaches that preserve physiologically relevant protein interactions:

• Affinity purification coupled with mass spectrometry (AP-MS): Using NDUFA3 as bait with either epitope tags or specific antibodies, followed by mass spectrometric identification of co-purified proteins. This approach has successfully identified interactions between Complex I subunits.

• Proximity labeling methods: Techniques such as BioID or APEX, where NDUFA3 is fused to a biotin ligase or peroxidase, enable biotinylation of proximal proteins that can then be identified by mass spectrometry. These approaches are particularly valuable for capturing transient interactions.

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinking of protein complexes followed by MS analysis can identify specific interaction interfaces between NDUFA3 and other mitochondrial proteins, providing structural insights into these associations.

• Blue Native PAGE: This technique preserves native protein complexes and can assess NDUFA3 incorporation into different subcomplexes of Complex I, revealing assembly intermediates or alternative complex formations.

• Förster Resonance Energy Transfer (FRET): For studying interactions in living cells, FRET between fluorophore-tagged NDUFA3 and potential interaction partners provides spatial and temporal information about these associations.

When interpreting interaction data, researchers should consider the dynamic nature of mitochondrial protein complexes and validate key interactions using multiple complementary techniques to distinguish genuine biological interactions from technical artifacts.

How can researchers effectively measure NDUFA3 expression and activity in different experimental systems?

Comprehensive assessment of NDUFA3 expression and activity requires multilevel analysis spanning from gene expression to functional consequences:

• Transcriptional analysis:

  • Quantitative RT-PCR with primers specific to NDUFA3

  • RNA-Seq for transcriptome-wide context of NDUFA3 expression changes

  • Consideration of HDAC/H3K27ac-mediated transcriptional regulation

• Protein expression quantification:

  • Western blot analysis with validated NDUFA3-specific antibodies

  • Immunocytochemistry/immunohistochemistry for subcellular localization

  • Targeted proteomics using selected reaction monitoring (SRM) for absolute quantification

• Functional activity assessment:

  • Complex I enzymatic activity assays measuring NADH oxidation

  • Oxygen consumption rate (OCR) using platforms like Seahorse XF analyzer

  • Mitochondrial membrane potential (MMP) using fluorescent indicators like JC-1

  • ROS production measurement using fluorescent probes like DCFDA

• Experimental models comparison:

  • Wild-type baseline conditions

  • NDUFA3 knockout or knockdown conditions

  • NDUFA3 overexpression systems

  • Treatment with mitochondrial inhibitors like rotenone as positive controls

This multilevel approach provides comprehensive insights into NDUFA3 regulation and function across different experimental systems, enabling robust cross-validation of findings and identification of context-specific effects.

How should researchers interpret contradictory findings regarding NDUFA3 function across different model systems?

When encountering contradictory findings regarding NDUFA3 function across different model systems, researchers should systematically evaluate several potential sources of discrepancy:

• Species-specific differences: Evolutionary analyses have demonstrated variation in mitochondrial genes across species, with different patterns of nucleotide substitutions . These differences may manifest as functional variations in NDUFA3 behavior across species-derived models.

• Cell type specificity: NDUFA3 function and regulation may vary considerably across different cell types due to:

  • Differences in mitochondrial content and morphology

  • Tissue-specific energy demands and metabolic profiles

  • Varying expression of other Complex I components and regulatory factors

• Experimental conditions: Critical parameters that may drive contradictory results include:

  • Glucose concentration (normal versus high glucose conditions show markedly different NDUFA3 effects)

  • Oxygen tension during cell culture

  • Presence of mitochondrial stressors or inhibitors

  • Duration of NDUFA3 manipulation (acute versus chronic)

• Methodological variations: Different techniques for measuring the same parameter (e.g., various ROS detection methods) may yield different results based on their specific sensitivities and limitations.

To reconcile contradictory findings, researchers should perform side-by-side comparisons using standardized protocols across different model systems, explicitly controlling for these variables. Meta-analyses and systematic reviews, with careful attention to methodological details, can help identify patterns that explain apparent contradictions.

What common technical challenges arise in NDUFA3 research and how can they be effectively addressed?

NDUFA3 research presents several technical challenges that require specific troubleshooting approaches:

• Protein solubility and stability:

  • Challenge: As a mitochondrial membrane protein, NDUFA3 can be difficult to maintain in solution

  • Solution: Optimize buffer conditions (using Tris/PBS with 6% Trehalose at pH 8.0) ; consider membrane mimetics for functional studies

• Knockout/knockdown lethality:

  • Challenge: Complete NDUFA3 loss may be lethal due to its essential role in energy metabolism

  • Solution: Implement inducible or partial knockdown systems; use tissue-specific knockouts in animal models

• Functional compensation mechanisms:

  • Challenge: Cells may upregulate alternative pathways following NDUFA3 manipulation

  • Solution: Employ acute manipulation systems; analyze early timepoints; consider combinatorial approaches targeting multiple pathways

• Mitochondrial assay variability:

  • Challenge: Measurements of mitochondrial function (OCR, ROS, MMP) often show high variability

  • Solution: Increase biological and technical replicates; include internal standards; normalize to appropriate controls; standardize cell confluency and passage number

• Recombinant protein activity verification:

  • Challenge: Ensuring recombinant NDUFA3 retains native function

  • Solution: Perform rescue experiments in knockout models; compare activity with endogenous protein; validate complex I incorporation

• Experimental result interpretation:

  • Challenge: Distinguishing direct NDUFA3 effects from secondary consequences

  • Solution: Include appropriate controls (e.g., rotenone treatment) ; perform time-course studies; use multiple complementary assays

Addressing these challenges through rigorous experimental design and appropriate controls ensures more reliable and interpretable results in NDUFA3 research.

How can researchers differentiate between direct effects of NDUFA3 manipulation and secondary consequences of mitochondrial dysfunction?

Differentiating direct NDUFA3 effects from secondary mitochondrial dysfunction consequences requires a systematic experimental approach:

• Temporal analysis: Monitoring changes following NDUFA3 manipulation across multiple timepoints helps establish the sequence of events. Primary effects typically manifest earlier than secondary consequences.

• Dose-response relationships: Varying the degree of NDUFA3 manipulation (partial versus complete knockdown, different overexpression levels) can reveal threshold effects that distinguish direct from indirect consequences.

• Rescue experiments: Re-introducing wild-type NDUFA3 in knockout models should reverse direct effects, while some secondary adaptations may persist. Similarly, introducing functionally deficient NDUFA3 mutants can help identify structure-function relationships.

• Comparative analysis with known mitochondrial inhibitors: Side-by-side comparison with specific inhibitors like:

  • Rotenone (Complex I inhibitor)

  • Antimycin A (Complex III inhibitor)

  • Oligomycin (ATP synthase inhibitor)

  • CCCP (mitochondrial uncoupler)

• Pathway inhibition studies: Using specific inhibitors of downstream pathways potentially activated by mitochondrial dysfunction (e.g., ROS scavengers, caspase inhibitors) can help distinguish which effects are secondary to these pathways versus directly linked to NDUFA3.

• Multi-omics approach: Integrating transcriptomics, proteomics, and metabolomics data following NDUFA3 manipulation can distinguish direct molecular interactions from broader adaptive responses through network analysis.

This multifaceted approach enables researchers to construct a hierarchical model of NDUFA3 functions, distinguishing its direct molecular roles from the cascade of secondary adaptations to mitochondrial dysfunction.

How might NDUFA3 research inform therapeutic approaches for mitochondrial dysfunction in metabolic disorders?

NDUFA3 research offers several promising directions for therapeutic development targeting mitochondrial dysfunction in metabolic disorders:

• Gene therapy approaches: The protective effects observed with NDUFA3 overexpression in high glucose conditions suggest that delivering functional NDUFA3 could mitigate diabetes-related cellular injuries. Adeno-associated virus (AAV) vectors could be engineered to target tissues most affected by mitochondrial dysfunction in metabolic disorders.

• Epigenetic modulation: The involvement of HDAC/H3K27ac mechanisms in regulating NDUFA3 transcription indicates that epigenetic drugs such as specific HDAC inhibitors could potentially increase endogenous NDUFA3 expression, enhancing mitochondrial function in metabolic disorders.

• Small molecule stabilizers: Developing compounds that stabilize NDUFA3 or enhance its incorporation into Complex I could improve mitochondrial function in conditions where NDUFA3 is present but dysfunctional due to metabolic stress.

• Mitochondrial-targeted antioxidants: Since NDUFA3 dysfunction contributes to increased ROS production under high glucose conditions , mitochondrial-targeted antioxidants could complement NDUFA3-focused therapies by mitigating downstream oxidative damage.

• Metabolic pathway modulators: Compounds that reduce the metabolic burden on compromised mitochondria or promote alternative energy production pathways could provide symptomatic relief while more direct NDUFA3-targeting approaches are developed.

These therapeutic strategies require careful development with consideration of tissue-specific effects, potential off-target consequences, and effective methods for targeting mitochondria in relevant tissues affected by metabolic disorders.

What insights can comparative studies of NDUFA3 across primate species provide for human disease understanding?

Comparative studies of NDUFA3 across primate species offer valuable insights into human disease mechanisms and potential evolutionary adaptations in mitochondrial function:

• Identification of functionally critical regions: Sequences conserved across primates likely represent domains essential for NDUFA3 function, while variable regions may contribute to species-specific adaptations. This information helps predict which human NDUFA3 mutations are likely pathogenic.

• Natural experiments in adaptation: The observation that mitochondrial genes like NDUFA3 show patterns of substitution consistent with slightly deleterious effects suggests an evolutionary mechanism for maintaining mitochondrial function despite genetic variation.

• Species-specific metabolic adaptations: Differences in NDUFA3 sequence between species may reflect adaptations to different metabolic demands or environmental pressures, potentially informing therapeutic approaches that mimic beneficial adaptations.

• Disease susceptibility variations: Comparing human disease-associated NDUFA3 variants with corresponding positions in other primates can reveal whether certain species have naturally evolved protective mechanisms against mitochondrial dysfunction.

• Evolutionary trade-offs: Understanding how variations in NDUFA3 across primates balance energy production efficiency against ROS generation could inform therapeutic strategies aimed at optimizing this balance in human disease states.

These comparative insights contribute to a deeper understanding of mitochondrial biology evolution and may inspire biomimetic approaches to treating human mitochondrial disorders.