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

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

NDUFB3 is an accessory subunit of mitochondrial Complex I, which forms part of the hydrophobic membrane arm within subcomplex Iβ. This protein plays a crucial role in the assembly and stability of Complex I, as demonstrated by assembly profile analysis using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE). When NDUFB3 is mutated or absent, partially assembled Complex I intermediates of approximately 650 kDa can be observed, indicating its importance in the complete assembly of the functional complex .

Methodologically, researchers investigating NDUFB3's structural role should employ:

• BN-PAGE analysis to assess Complex I assembly

• SDS-PAGE followed by immunoblotting with antibodies against various Complex I subunits

• Respirometry measurements to assess functional consequences of NDUFB3 modifications

How conserved is NDUFB3 across species, particularly between humans and Pan troglodytes?

The NDUFB3 protein contains evolutionarily conserved amino acid residues, particularly in functional domains. Specific residues like Trp22 show high conservation across species, explaining why mutations at these positions often result in pathological conditions . When conducting comparative studies between human and Pan troglodytes NDUFB3, researchers should:

• Perform multiple sequence alignment using tools like Clustal Omega or MUSCLE

• Calculate sequence identity and similarity percentages

• Create phylogenetic trees to visualize evolutionary relationships

• Focus particular attention on functional domains and critical residues

What methods are most effective for recombinant expression of Pan troglodytes NDUFB3?

For optimal recombinant expression of Pan troglodytes NDUFB3:

• Select an appropriate expression system:

  • Bacterial systems (E. coli) may be suitable for structural studies but may lack proper post-translational modifications

  • Insect cell systems (Sf9, Hi5) often provide better folding for mitochondrial proteins

  • Mammalian expression systems offer the most native-like post-translational modifications

• Codon optimization:

  • Adapt the Pan troglodytes NDUFB3 sequence to the codon usage of your expression system

  • Consider using commercially available codon optimization algorithms

• Purification strategy:

  • Add appropriate affinity tags (His6, GST, FLAG) that won't interfere with protein function

  • Include protease cleavage sites to remove tags if necessary for functional studies

  • Develop a multi-step purification protocol (affinity chromatography followed by size exclusion)

• Validation:

  • Confirm expression and purification by SDS-PAGE and western blotting

  • Verify protein folding using circular dichroism spectroscopy

  • Assess functional activity in reconstitution assays

How do mutations in Pan troglodytes NDUFB3 affect Complex I assembly and function compared to human mutations?

Mutations in NDUFB3 have been shown to significantly impact Complex I assembly and function in humans. The p.Trp22Arg variant in humans results in decreased steady-state levels of Complex I subunit proteins NDUFB8 and NDUFA9, as well as impaired assembly of the complete Complex I . When investigating the effects of similar mutations in Pan troglodytes NDUFB3, researchers should:

• Generate equivalent mutations in Pan troglodytes NDUFB3 using site-directed mutagenesis

• Express wild-type and mutant proteins in appropriate cell models

• Assess Complex I assembly using BN-PAGE analysis

• Measure protein stability and half-life using cycloheximide chase assays

• Quantify Complex I activity using spectrophotometric assays for NADH:ubiquinone oxidoreductase

• Measure oxygen consumption rates using high-resolution respirometry

• Compare results with equivalent human mutations to identify species-specific differences

What is the role of NDUFB3 in regulating mitochondrial reactive oxygen species (mitoROS) production in Pan troglodytes cells?

NDUFB3 has been identified as a regulator of mitochondrial reactive oxygen species (mitoROS). Research in human cells has shown that NDUFB3 knockdown significantly reduces mitoROS levels, while overexpression increases mitoROS production . To investigate this role in Pan troglodytes cells:

• Establish Pan troglodytes cell lines with NDUFB3 knockdown and overexpression

• Measure mitoROS levels using:

  • Flow cytometry with MitoSOX staining

  • Plate-based fluorescence assays with cellular ROS-sensitive dyes

  • EPR spectroscopy for highly precise quantification

• Assess mitochondrial function parameters, including:

  • Oxygen consumption rate

  • ATP production

  • Complex I activity

  • Mitochondrial membrane potential

• Compare results with equivalent human cell models to identify potential species-specific differences in NDUFB3 function

How can researchers effectively design experiments to study NDUFB3 involvement in mitochondrial disease models?

To effectively design experiments studying NDUFB3 in mitochondrial disease models:

• Model selection considerations:

  • Cell lines: Choose between established cell lines and patient-derived primary cells

  • Animal models: Consider developing Pan troglodytes NDUFB3 knockin/knockout models in mice

  • iPSC-derived models: Generate disease-relevant cell types from patient samples

• Experimental design framework:

  • Include appropriate controls (isogenic controls for genetic studies)

  • Plan time-course experiments to capture disease progression

  • Use multiple complementary techniques to assess mitochondrial function

• Key assays to include:

  • BN-PAGE and immunoblotting to assess Complex I assembly

  • High-resolution respirometry to measure oxygen consumption

  • ATP production assays

  • Mitochondrial membrane potential measurements

  • mitoROS quantification

  • Mitochondrial morphology analysis using confocal microscopy

• Data analysis approaches:

  • Employ statistical methods appropriate for the experimental design

  • Consider using systems biology approaches to integrate multiple data types

What techniques are most suitable for studying the interactions between NDUFB3 and other Complex I subunits?

To effectively study the protein-protein interactions of NDUFB3 with other Complex I subunits:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against NDUFB3 or epitope tags

  • Perform under mild detergent conditions to preserve interactions

  • Validate with reciprocal Co-IPs

• Proximity labeling approaches:

  • BioID: Fuse NDUFB3 with a biotin ligase (BirA*) to biotinylate proximal proteins

  • APEX2: Fuse NDUFB3 with APEX2 enzyme for proximity-based biotinylation

  • Analyze biotinylated proteins by mass spectrometry

• Crosslinking mass spectrometry (XL-MS):

  • Apply chemical crosslinkers to stabilize transient interactions

  • Digest crosslinked complexes and analyze by LC-MS/MS

  • Use specialized software to identify crosslinked peptides

• Cryo-electron microscopy:

  • Purify intact Complex I for structural analysis

  • Generate 3D reconstructions to visualize NDUFB3 interactions

  • Compare structures with and without NDUFB3 or with mutant variants

• Fluorescence-based techniques:

  • FRET (Förster Resonance Energy Transfer) for analyzing protein proximity

  • BiFC (Bimolecular Fluorescence Complementation) to visualize interactions in living cells

What are the optimal protocols for isolating functional mitochondria to study NDUFB3 in Pan troglodytes cells?

For isolating functional mitochondria from Pan troglodytes cells:

• Cell preparation:

  • Culture cells to 80-90% confluence

  • Wash with ice-cold PBS

  • Harvest by gentle scraping or trypsinization

• Isolation procedure:

  • Homogenize cells in isolation buffer (225 mM mannitol, 75 mM sucrose, 10 mM HEPES, 1 mM EGTA, pH 7.4)

  • Perform differential centrifugation:

    • 1,000g for 10 minutes to remove nuclei and unbroken cells

    • 10,000g for 15 minutes to pellet mitochondria

  • Purify further using Percoll gradient centrifugation if needed

• Quality assessment:

  • Measure respiratory control ratio using oxygen electrode

  • Assess membrane potential using fluorescent dyes (TMRM, JC-1)

  • Confirm mitochondrial marker proteins by immunoblotting (VDAC, TOM20)

  • Test for contamination with other cellular compartments

• Storage:

  • Use immediately for functional studies

  • For protein analysis, snap-freeze in liquid nitrogen and store at -80°C

How can researchers accurately measure Complex I activity in systems with modified NDUFB3?

To accurately measure Complex I activity in systems with modified NDUFB3:

• Spectrophotometric assays:

  • NADH:ubiquinone oxidoreductase activity:

    • Monitor NADH oxidation at 340 nm

    • Use specific Complex I inhibitors (rotenone) as controls

    • Calculate activity as rotenone-sensitive NADH oxidation rate

  • Diphenyleneiodonium (DPI)-sensitive NADH dehydrogenase activity

• High-resolution respirometry:

  • Measure oxygen consumption in intact cells or isolated mitochondria

  • Use substrate-uncoupler-inhibitor titration protocols:

    • Glutamate/malate or pyruvate/malate as Complex I substrates

    • ADP to stimulate oxidative phosphorylation

    • FCCP to assess maximal respiratory capacity

    • Rotenone to inhibit Complex I specifically

• In-gel activity assays:

  • Separate respiratory complexes by BN-PAGE

  • Incubate gels with NADH and nitrotetrazolium blue (NBT)

  • Quantify Complex I activity by densitometry of purple formazan bands

• Seahorse XF analysis:

  • Measure oxygen consumption rate (OCR) in live cells

  • Design assays with selective Complex I substrates and inhibitors

  • Calculate Complex I-dependent respiration

What are the best approaches for studying the impact of NDUFB3 variants on mitochondrial ROS production?

The following approaches are optimal for studying how NDUFB3 variants affect mitochondrial ROS production:

• Flow cytometry with fluorescent probes:

  • MitoSOX Red for mitochondrial superoxide detection

  • CM-H2DCFDA for general cellular ROS

  • Protocol should include appropriate controls and standardization

• Live-cell imaging:

  • Use confocal microscopy with ROS-sensitive fluorescent probes

  • Perform time-lapse imaging to monitor dynamic changes

  • Co-stain with mitochondrial markers to confirm localization

• Plate-based fluorescence assays:

  • Higher throughput than microscopy or flow cytometry

  • Suitable for screening multiple conditions

  • Less specific for mitochondrial vs. cytosolic ROS

• Electron Paramagnetic Resonance (EPR) spectroscopy:

  • Gold standard for ROS detection

  • Use spin traps or spin probes for specific ROS types

  • Provides quantitative measurements

• Oxidative damage markers:

  • Measure 8-oxoguanine, protein carbonylation, or lipid peroxidation

  • Serves as functional readout of ROS effects

  • Can be quantified by ELISA, immunoblotting, or mass spectrometry

• Antioxidant enzyme activities:

  • Measure SOD, catalase, glutathione peroxidase activities

  • Assess cellular response to altered ROS levels

  • Complement direct ROS measurements

How should researchers analyze and interpret contradictory data regarding NDUFB3 function?

When faced with contradictory data regarding NDUFB3 function:

• Systematic approach to resolving contradictions:

  • Compare experimental methodologies in detail

  • Assess cell types and model systems used

  • Examine genetic backgrounds and potential compensatory mechanisms

  • Consider environmental conditions (culture media, oxygen levels)

• Statistical considerations:

  • Evaluate statistical power of each study

  • Look for potential outliers or biased data points

  • Consider whether appropriate statistical tests were applied

  • Meta-analysis approaches may help reconcile disparate findings

• Technical validation:

  • Replicate key experiments using multiple complementary techniques

  • Validate antibodies and reagents rigorously

  • Use genetic rescue experiments to confirm specificity

  • Consider blind experimental design to minimize bias

• Biological context:

  • Tissue-specific or cell type-specific effects may explain differences

  • Developmental timing may influence results

  • Consider potential species differences when comparing human and Pan troglodytes data

  • Evaluate whether contradictory findings relate to primary or secondary effects

What bioinformatic tools are most appropriate for analyzing NDUFB3 sequence variants and their impact?

For analyzing NDUFB3 sequence variants and their potential impact:

• Sequence conservation analysis:

  • Tools: ConSurf, Clustal Omega, MUSCLE

  • Purpose: Identify evolutionarily conserved residues likely to be functionally important

  • Output: Conservation scores that can be mapped to protein structure

• Variant effect prediction:

  • Tools: SIFT, PolyPhen-2, PROVEAN, MutationTaster

  • Purpose: Predict functional impact of amino acid substitutions

  • Integration: Combine multiple predictors for consensus scoring

• Protein structure analysis:

  • Tools: PyMOL, SWISS-MODEL, AlphaFold

  • Purpose: Model variant effects on protein structure and interactions

  • Applications: Visualize location of variants in 3D structure, predict stability changes

• Population frequency analysis:

  • Databases: gnomAD, ExAC, 1000 Genomes

  • Purpose: Determine rarity of variants in population

  • Context: Rare variants in conserved regions are more likely pathogenic

• Phylogenetic analysis:

  • Tools: MEGA, RAxML, MrBayes

  • Purpose: Understand evolutionary relationships and conservation

  • Applications: Identify lineage-specific constraints and adaptations

What are the most promising approaches for therapeutic targeting of NDUFB3-related mitochondrial dysfunction?

Several promising therapeutic approaches for NDUFB3-related mitochondrial dysfunction include:

• Gene therapy strategies:

  • AAV-mediated gene delivery of wild-type NDUFB3

  • CRISPR-Cas9 gene editing to correct pathogenic mutations

  • Challenges include delivery to mitochondria and tissue specificity

• Small molecule approaches:

  • Complex I bypass strategies using alternative electron carriers

  • Compounds that stabilize partially assembled Complex I

  • Antioxidants targeting mitochondrial ROS production

• Mitochondrial transplantation:

  • Direct transfer of healthy mitochondria to cells with dysfunctional NDUFB3

  • Methodological considerations include isolation of donor mitochondria and delivery methods

  • Preliminary success in cardiac and other tissues

• Metabolic bypass strategies:

  • Dietary interventions (ketogenic diet)

  • Metabolites that can enter electron transport chain downstream of Complex I

  • Methodological assessment using cellular and animal models

• Mitochondrial biogenesis stimulation:

  • PGC-1α activators to increase mitochondrial mass

  • NAD+ precursors (NMN, NR) to enhance mitochondrial function

  • Exercise mimetics to stimulate physiological adaptations

How might comparative studies between human and Pan troglodytes NDUFB3 advance our understanding of mitochondrial evolution?

Comparative studies between human and Pan troglodytes NDUFB3 can provide valuable insights into mitochondrial evolution:

• Research framework:

  • Compare sequence, structure, and function systematically

  • Examine species-specific differences in interaction networks

  • Study adaptive changes related to metabolic requirements

• Key methodological approaches:

  • Reciprocal complementation experiments to test functional conservation

  • Creation of chimeric proteins to map species-specific functional domains

  • Comparative proteomics of Complex I composition and assembly

• Evolutionary context analysis:

  • Study selective pressures using dN/dS ratios

  • Identify lineage-specific accelerated evolution

  • Correlate with ecological and physiological differences

• Specific research questions to address:

  • Do human-specific NDUFB3 features relate to brain energy metabolism?

  • Have dietary differences driven adaptive changes?

  • Do differences in lifespan correlate with NDUFB3 function?

• Broader implications:

  • Insights into human-specific mitochondrial adaptations

  • Understanding of fundamental mechanisms of protein evolution

  • Potential applications to human disease understanding