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

What is the structural and functional role of NDUFB3 in mitochondrial complex I?

NDUFB3 serves as an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), which is the first enzyme in the electron transport chain. Unlike core subunits, NDUFB3 is believed not to be directly involved in catalysis but plays critical roles in complex assembly and stability .

Complex I functions in transferring electrons from NADH to the respiratory chain, with ubiquinone serving as the immediate electron acceptor . NDUFB3 is positioned within subcomplex Iβ of the hydrophobic membrane arm of Complex I . When examining the function of NDUFB3, it's important to note that complementation experiments with wild-type NDUFB3 cDNA can restore both complex I activity and assembly in deficient cells, demonstrating its essential role in maintaining proper complex I structure and function .

Experimental approach: To study NDUFB3's role in complex I assembly, researchers commonly use blue-native PAGE (BN-PAGE) separation followed by immunoblotting with antibodies against complex I subunits such as NDUFA9 . Two-dimensional BN/SDS-PAGE separation combined with fluorescent labeling can quantify the impact of NDUFB3 mutations on complex I assembly and supercomplexes formation .

What methods are most effective for measuring NDUFB3 expression in biological samples?

Several complementary techniques can be employed to comprehensively assess NDUFB3 expression:

• Database mining: GEPIA and Human Protein Atlas (HPA) databases provide valuable resources for studying expression characteristics of NDUFB3 across tissues and disease states .

• Protein detection methods:

  • Western blotting using specific antibodies against NDUFB3

  • Immunohistochemistry for tissue localization

  • Fluorescent labeling combined with BN-PAGE for complex I assembly analysis

• Gene expression analysis:

  • RT-qPCR for mRNA quantification

  • RNA-Seq for transcriptomic profiling

  • Nanostring platform for precise quantification in clinical samples

Methodological consideration: When analyzing NDUFB3 expression in disease contexts, it's important to normalize expression levels to appropriate reference genes and include multiple tissue or cell types for comparison. For example, a study of NDUFB3 in gynecological cancers compared expression across ovarian cancer (OV), uterine corpus endometrial carcinoma (UCEC), and cervical squamous cell carcinoma (CESC) to identify tissue-specific patterns .

Mutations in NDUFB3 are associated with mitochondrial complex I deficiency (particularly nuclear type 25) and several distinctive clinical features:

• Intrauterine growth restriction (IUGR)

• Short stature (height <9th centile)

• Characteristic facial features including a prominent forehead, smooth philtrum, deep-set eyes, and low-set ears

• Variable neurological manifestations

• Metabolic dysfunction, including lactic acidosis in some cases

The p.Trp22Arg variant in NDUFB3 represents a recurrent mutation identified particularly in patients of Irish ancestry and is associated with a relatively good long-term prognosis despite initial metabolic crisis .

Research approach: When studying NDUFB3-related diseases, clinical phenotyping should include careful assessment of growth parameters, dysmorphic features, and metabolic function. Recognition of the distinctive facial features associated with the p.Trp22Arg variant can facilitate targeted genetic testing without requiring muscle biopsy .

How does NDUFB3 influence reactive oxygen species (ROS) production and what experimental approaches best measure this relationship?

NDUFB3 plays a critical role in regulating mitochondrial ROS production through its function in complex I. Research has demonstrated that:

• NDUFB3 knockdown significantly reduces mitochondrial ROS (mitoROS) levels in thyroid cancer cell lines (BCPAP and C643) .

• In gynecological tumor cells, NDUFB3 depletion increases intracellular ROS production, inducing cell cycle arrest and apoptosis . This seemingly contradictory finding suggests context-dependent effects.

• NDUFB3 expression correlates with mitoROS levels, with lower expression predicting poor clinical outcomes in thyroid cancer patients .

Methodological approaches for measuring ROS:

• Dihydroethidium fluorescent probe: Used to detect ROS production in NDUFB3-depleted tumor cells .

• MitoSOX Red: Specific for mitochondrial superoxide detection.

• DCFDA assay: Measures general cellular ROS levels.

• Genetic ROS reporters: Such as HyPer or roGFP for real-time monitoring.

Experimental design considerations: When investigating NDUFB3's impact on ROS, researchers should:

• Include appropriate positive controls (e.g., antimycin A for complex III-derived ROS)

• Validate findings with multiple ROS detection methods

• Consider the kinetics of ROS production following NDUFB3 manipulation

• Examine the interplay between ROS and other mitochondrial parameters (membrane potential, ATP production)

What is the role of NDUFB3 in cancer progression and what therapeutic implications does this have?

NDUFB3 demonstrates context-dependent roles in cancer progression with significant implications for targeted therapies:

• Gynecological cancers:

  • NDUFB3 is highly expressed in ovarian cancer (OV), uterine corpus endometrial carcinoma (UCEC), and cervical squamous cell carcinoma (CESC) .

  • Knocking down NDUFB3 inhibits proliferation of CESC, OV, and UCEC cells .

  • NDUFB3 expression is associated with multiple immunomodulators in these cancers .

  • NDUFB3 is predicted to modulate MAPK signaling pathways in gynecological tumors .

• Thyroid cancer:

  • Lower expression of NDUFB3 is associated with poor clinical outcomes .

  • NDUFB3 overexpression increases mitochondrial functions, including oxygen consumption rate, ATP levels, complex I activity, and mitoROS levels .

• Therapeutic targeting:

  • Wedelolactone has been identified as a potential small molecule inhibitor that binds to the active pocket of NDUFB3 .

  • Wedelolactone demonstrates cytotoxicity against CESC, OV, and UCEC cells partly via NDUFB3 inhibition .

Experimental approaches for cancer studies:

• Cell proliferation: CCK-8 assay following NDUFB3 depletion or wedelolactone treatment .

• Virtual screening and molecular docking: To identify compounds targeting NDUFB3 .

• Cell cycle and apoptosis: Flow cytometry to evaluate the cellular response to NDUFB3 modulation .

• Pathway analysis: GO and KEGG enrichment analyses using R software clusterProfiler package .

How can genetic perturbation experiments be designed to elucidate NDUFB3 function in complex biological systems?

Designing effective genetic perturbation experiments for NDUFB3 requires careful planning and consideration of multiple approaches:

• CRISPR-Cas9 genome editing:

  • Complete knockout to assess essential functions

  • Knock-in of specific mutations (e.g., p.Trp22Arg) to recapitulate patient phenotypes

  • Base editing for precise nucleotide changes

• RNAi approaches:

  • siRNA for transient knockdown to assess acute effects

  • shRNA for stable knockdown to study long-term consequences

• Overexpression systems:

  • Wild-type NDUFB3 for complementation studies

  • Mutant variants to assess dominant-negative effects

The BioDiscoveryAgent approach described in search result represents an AI-assisted method for designing genetic perturbation experiments that could be applied to NDUFB3 research. This approach:

• Leverages prior biological knowledge

• Designs experimental batches that prioritize genes likely to exhibit desired phenotypes

• Allows for iterative refinement based on experimental results

Experimental design considerations:

• Include appropriate controls (wild-type, empty vector)

• Validate knockdown/overexpression at both mRNA and protein levels

• Assess phenotypes across multiple cellular functions (growth, metabolism, ROS production)

• Consider combinatorial perturbations with other complex I subunits

How does NDUFB3 contribute to mitochondrial complex I assembly and what methodologies best assess assembly defects?

NDUFB3 plays a critical role in complex I assembly and stability, with mutations resulting in characteristic assembly defects:

• Assembly process involvement:

  • NDUFB3 is part of subcomplex Iβ of the hydrophobic membrane arm .

  • Loss of NDUFB3 function impairs assembly or stability of the entire complex I.

  • Wild-type NDUFB3 expression rescues both complex I activity and assembly in patient fibroblasts .

• Assembly defect characteristics:

  • Reduced amounts of fully assembled complex I

  • Impaired formation of supercomplexes

  • Accumulation of partially assembled intermediates of ~650 kDa, consistent with defects in subcomplex Iβ .

Methodological approaches for assessing assembly:

• Blue-native PAGE (BN-PAGE):

  • One-dimensional BN-PAGE to visualize intact complexes

  • Two-dimensional BN/SDS-PAGE to identify specific subunits

  • Quantification of fluorescein-labeled mitochondrial complexes

• Western blotting with antibodies against:

  • NDUFB3 itself

  • Other complex I subunits (NDUFA9, NDUFB8, NDUFS3)

  • Subunits of other OXPHOS complexes as controls

• Complementation studies:

  • Expression of wild-type NDUFB3 cDNA in patient fibroblasts

  • Assessment of rescue of complex I assembly and function

Quantitative analysis: The complete restoration of complex I following wild-type NDUFB3 expression provides strong evidence of its essential role in complex assembly. Research has shown that transduction with wild-type NDUFB3 can increase mitochondrial supercomplex formation up to 43% of control levels in patient fibroblasts .

What is known about the potential role of NDUFB3 in inflammatory conditions such as sepsis?

Recent research has identified NDUFB3 as a potential biomarker and therapeutic target in sepsis:

• Expression patterns:

  • NDUFB3 was identified as one of three differentially expressed mitochondria-related genes (DE-MiRGs) in sepsis .

  • Significantly elevated expression levels of NDUFB3 were confirmed in LPS-stimulated sepsis models .

• Functional implications:

  • NDUFB3 contributes to mitochondrial quality imbalance in sepsis .

  • NDUFB3 expression shows negative correlation with LETMD1 and positive correlation with BCKDHB in sepsis patients .

• Immune system interactions:

  • NDUFB3 may influence immune cell infiltration in sepsis .

  • Analysis of sepsis patients revealed significant differences in the immune microenvironment landscape compared to healthy controls .

Experimental approaches:

• WGCNA (Weighted Gene Co-expression Network Analysis) and machine learning algorithms (Random Forest and LASSO) to identify feature biomarkers .

• CIBERSORT algorithm to assess immune cell infiltration patterns .

• In vitro experiments and confocal microscopy to confirm expression changes .

Research implications: The identification of NDUFB3 as a potential therapeutic target in sepsis opens new avenues for intervention strategies targeting mitochondrial function. Further research is needed to elucidate the precise mechanisms by which NDUFB3 contributes to sepsis pathogenesis and whether modulation of its activity could provide therapeutic benefit.

What approaches can be used to identify small molecule modulators of NDUFB3 function and assess their therapeutic potential?

Identifying and validating small molecule modulators of NDUFB3 requires a multifaceted approach:

• Virtual screening and molecular docking:

  • Used successfully to identify wedelolactone as a potential NDUFB3 inhibitor .

  • Screens natural compounds for binding to the active pocket of NDUFB3.

• In vitro validation:

  • Cell viability assays (CCK-8) to assess cytotoxicity .

  • Complex I activity measurements to confirm target engagement.

  • ROS detection using fluorescent probes to monitor functional effects .

• Structure-activity relationship studies:

  • Modification of lead compounds to improve potency and specificity.

  • Assessment of binding site interactions through modeling and mutagenesis.

• Therapeutic assessment:

  • Context-dependent evaluation (cancer cells vs. mitochondrial disease models).

  • Comparison with existing complex I modulators.

  • Evaluation in patient-derived cell models.

Case study: Wedelolactone
Wedelolactone was identified as a small molecule with strong binding affinity for the active pocket of NDUFB3. Research demonstrated that:

• It exerts cytotoxicity against gynecological cancer cells partly through NDUFB3 inhibition .

• It induces similar cellular effects to NDUFB3 knockdown, including increased ROS production and cell cycle arrest .

Methodological considerations:

• Include appropriate positive and negative controls in all assays.

• Validate target engagement using multiple orthogonal approaches.

• Assess off-target effects through profiling against other mitochondrial complexes.

• Evaluate pharmacokinetic properties for promising compounds.

What are the optimal conditions for expressing and purifying recombinant NDUFB3 for structural and functional studies?

Producing high-quality recombinant NDUFB3 presents several challenges due to its hydrophobic nature and role as a membrane protein. Based on available research and protein expression expertise:

Expression systems:

• E. coli: Successfully used for expressing NDUFB3, particularly with tags that enhance solubility such as GST .

• Mammalian cells: More suitable for studies requiring proper folding and post-translational modifications.

• Cell-free systems: May be advantageous for membrane proteins that are difficult to express in conventional systems.

Purification strategies:

• Affinity chromatography: Using N-terminal GST tag as demonstrated in commercial preparations .

• Size exclusion chromatography: For further purification after affinity steps.

• Detergent selection: Critical for maintaining native structure of membrane proteins.

Quality control assessments:

• SDS-PAGE and western blotting to confirm identity and purity

• Mass spectrometry for accurate mass determination

• Circular dichroism to assess secondary structure

• Functional assays to confirm biological activity

Methodological considerations:

• Expression temperature, induction conditions, and buffer composition must be optimized

• The addition of protease inhibitors is essential during purification

• For structural studies, consider incorporation into nanodiscs or amphipols to maintain native environment

How can mutations in NDUFB3 be functionally characterized to determine their pathogenicity?

A comprehensive approach to functionally characterizing NDUFB3 mutations includes:

• Complementation studies:

  • Expression of wild-type versus mutant NDUFB3 in patient fibroblasts

  • Assessment of complex I activity restoration

  • Measurement of complex I assembly using BN-PAGE

• Protein stability and expression:

  • Western blotting to assess steady-state levels of NDUFB3 and other complex I subunits

  • Pulse-chase experiments to determine protein half-life

  • Assessment of mRNA levels to distinguish translational from post-translational effects

• Complex I function measures:

  • NADH:ubiquinone oxidoreductase activity assays

  • Oxygen consumption rate measurements

  • ATP production capacity

Case study: p.Trp22Arg mutation
The p.Trp22Arg mutation in NDUFB3 has been extensively characterized:

• It fails to restore complex I activity when expressed in patient fibroblasts, unlike wild-type NDUFB3 .

• It results in reduced amounts of fully assembled complex I and impaired formation of supercomplexes .

• Despite its biochemical impact, patients with this mutation often have a better prognosis than expected .

Data interpretation:
When characterizing novel variants, compare complex I activity and assembly data with established pathogenic mutations like p.Trp22Arg. Rescue of complex I activity and assembly by wild-type but not mutant NDUFB3 provides strong evidence for pathogenicity .