Recombinant Pan troglodytes NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 5, mitochondrial (NDUFB5)

What is the biochemical function of NDUFB5 in mitochondrial respiration?

To methodologically assess NDUFB5 function, researchers should consider:

• Oxygen consumption rate measurements in cells with modulated NDUFB5 expression

• Blue native gel electrophoresis to examine complex I assembly integrity

• NADH oxidation rate assays with isolated mitochondria or purified complexes

• Mitochondrial membrane potential measurements using fluorescent probes

How is NDUFB5 expression regulated at the post-transcriptional level?

NDUFB5 expression is regulated through several post-transcriptional mechanisms, with m6A RNA modification emerging as particularly significant. Recent research demonstrates that methyltransferase-like 3 (METTL3) mediates m6A modification of NDUFB5 mRNA, which is subsequently recognized by insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) . This modification enhances NDUFB5 mRNA stability and translation efficiency.

To experimentally investigate NDUFB5 post-transcriptional regulation:

• mRNA stability can be assessed using actinomycin D chase experiments (0.2 mM for 0, 2, 4, and 6 hours) followed by qRT-PCR quantification

• NDUFB5 3'UTR luciferase reporter assays can evaluate regulatory element function

• m6A RNA immunoprecipitation (m6A-RIP) identifies specific methylation sites

• RNA-binding protein immunoprecipitation reveals protein-RNA interactions

What is the sequence homology of Pan troglodytes NDUFB5 compared to other species?

Pan troglodytes (chimpanzee) NDUFB5 demonstrates high sequence conservation across vertebrate species, reflecting its fundamental importance in mitochondrial function. Comparative analysis reveals the following homology percentages:

This high conservation across diverse species suggests evolutionary pressure to maintain NDUFB5 structure and function . Methodologically, researchers can conduct multiple sequence alignments to identify conserved domains and critical functional residues when designing experiments with recombinant NDUFB5.

What expression systems are optimal for producing functional recombinant Pan troglodytes NDUFB5?

The production of functional recombinant Pan troglodytes NDUFB5 requires careful consideration of expression systems to ensure proper folding and activity. Based on its mitochondrial localization and interaction requirements, the following methodological approaches are recommended:

• Mammalian expression systems:

  • HEK293T cells provide appropriate post-translational modifications and chaperone systems

  • Use of mitochondrial targeting sequences improves subcellular localization

  • Inducible expression systems prevent potential toxicity from overexpression

• Insect cell/baculovirus systems:

  • High yield while maintaining proper protein folding

  • Suitable for structural studies requiring larger protein quantities

  • Less expensive than mammalian systems while providing eukaryotic modifications

• Optimization parameters:

  • Expression at lower temperatures (16-25°C) improves folding efficiency

  • Addition of solubility tags (SUMO, MBP) increases soluble protein yield

  • Codon optimization for the selected host improves translation efficiency

  • Inclusion of protease inhibitors throughout purification prevents degradation

The choice between these systems should be guided by the specific experimental requirements and downstream applications.

What are effective strategies for purifying recombinant NDUFB5 while maintaining its native conformation?

Purification of recombinant NDUFB5 presents challenges due to its hydrophobic nature and requirement for native conformation. Effective methodological approaches include:

• Membrane protein extraction:

  • Mild detergents (DDM, LMNG, or digitonin) preserve protein-protein interactions

  • Detergent screening to identify optimal solubilization conditions

  • Gradual detergent exchange during purification to maintain stability

• Chromatography strategy:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Ion exchange chromatography as a polishing step

• Stability considerations:

  • Buffer optimization including pH, salt concentration, and glycerol content

  • Addition of lipids or nanodiscs to mimic native membrane environment

  • Inclusion of reducing agents to maintain thiol groups in native states

Validation of purified protein should include functional assays measuring NADH oxidation activity and structural assessment through circular dichroism or limited proteolysis.

How can the successful incorporation of recombinant NDUFB5 into complex I be verified?

Verifying the successful incorporation of recombinant NDUFB5 into complex I is critical for functional studies. Methodological approaches include:

• Blue native polyacrylamide gel electrophoresis (BN-PAGE):

  • Detects assembled complex I containing recombinant NDUFB5

  • Allows identification of assembly intermediates

  • Can be followed by second-dimension SDS-PAGE to confirm subunit composition

• Immunological techniques:

  • Co-immunoprecipitation with antibodies against core complex I subunits

  • Western blotting of BN-PAGE gels using anti-NDUFB5 antibodies

  • Immunofluorescence microscopy to confirm mitochondrial localization

• Functional complementation:

  • Rescue experiments in NDUFB5-deficient cell lines

  • Restoration of complex I activity in knockout models

  • Oxygen consumption rate measurements before and after complementation

• Structural validation:

  • Cryo-electron microscopy of purified complex I

  • Crosslinking mass spectrometry to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to assess incorporation

These complementary approaches provide robust verification of successful NDUFB5 incorporation into functional complex I.

What experimental approaches effectively measure NDUFB5 contribution to mitochondrial respiration?

To quantify NDUFB5's specific contribution to mitochondrial respiration, researchers should employ multiple complementary techniques:

• Respirometry assays:

  • High-resolution respirometry using Oroboros or Seahorse platforms

  • Measurement of oxygen consumption rates with various substrates

  • Analysis of respiration in permeabilized cells versus isolated mitochondria

  • Comparison between wild-type and NDUFB5-deficient systems

• Complex I activity measurements:

  • Spectrophotometric assays tracking NADH oxidation rates

  • Rotenone-sensitive activity to distinguish complex I-specific function

  • In-gel activity staining following BN-PAGE separation

  • Site-specific electron transfer rate measurements

• Mitochondrial function parameters:

  • Membrane potential assessments using potentiometric dyes

  • ATP production capacity under different substrate conditions

  • Reactive oxygen species generation measurements

  • Mitochondrial calcium handling capacity

When interpreting these measurements, it's important to consider NDUFB5's role in the context of:

• Cell type-specific respiratory requirements

• Compensatory mechanisms in chronic versus acute NDUFB5 depletion

• Metabolic state of the cells (glycolytic versus oxidative)

• Presence of mitochondrial fusion promoters like M1, which can compensate for NDUFB5 deficiency

How does NDUFB5 affect cell viability and migration in experimental models?

NDUFB5 demonstrates significant effects on cellular functions beyond basic respiration. Research shows:

• Cell viability effects:

  • NDUFB5 overexpression promotes cell viability in AGEs-treated HUVECs

  • NDUFB5 knockdown reduces viability, which can be partially rescued by mitochondrial fusion promoter M1

  • The mechanism appears linked to maintenance of mitochondrial respiratory capacity

• Migration regulation:

  • NDUFB5 positively regulates cell migration in endothelial cell models

  • This function is particularly important in the context of wound healing

  • The migration phenotype correlates with mitochondrial respiration capacity

• Experimental protocols:

  • Cell viability assessment using MTT or similar metabolic assays

  • Wound healing scratch assays for migration quantification

  • Transwell migration assays for directional movement analysis

  • Real-time cell analysis systems for continuous monitoring

• In vivo correlates:

  • NDUFB5 promotes skin wound healing in diabetic mice

  • This suggests therapeutic potential in conditions with impaired tissue repair

These findings highlight NDUFB5's broader physiological roles beyond direct respiratory chain function and suggest potential applications in regenerative medicine research.

What methods are most effective for studying NDUFB5 interactions with other complex I subunits?

Understanding NDUFB5's interactions within complex I requires specialized techniques for membrane protein complexes:

• Crosslinking approaches:

  • Chemical crosslinking followed by mass spectrometry identification

  • Photo-reactive amino acid incorporation at specific positions

  • In vivo crosslinking in intact mitochondria

  • Distance constraint mapping from crosslink identification

• Proximity-based techniques:

  • BioID or APEX2 proximity labeling with NDUFB5 as the bait

  • Split fluorescent protein complementation assays

  • FRET or BRET approaches for dynamic interaction studies

  • Hydrogen-deuterium exchange mass spectrometry

• Structural biology methods:

  • Cryo-electron microscopy of intact complex I

  • X-ray crystallography of subcomplexes containing NDUFB5

  • AlphaFold or RoseTTAFold prediction validated by experimental data

  • Molecular dynamics simulations to predict interaction dynamics

• Genetic interaction mapping:

  • Synthetic lethality screening with other complex I subunits

  • Suppressor mutation analysis

  • Epistasis studies using double knockdown/knockout approaches

When designing these experiments, researchers should consider that NDUFB5 is a supernumerary subunit that may have both structural and regulatory functions within complex I.

How does NDUFB5 function contribute to diabetic wound healing mechanisms?

NDUFB5 plays a significant role in diabetic wound healing through several mechanisms:

• Endothelial cell function:

  • NDUFB5 promotes cell viability and migration in AGEs-treated HUVECs, which mimics diabetic conditions

  • These functions are critical for angiogenesis during wound repair

  • NDUFB5 maintains mitochondrial respiratory capacity under metabolic stress

• Experimental evidence:

  • In vitro models using HUVECs treated with various concentrations of AGEs (100, 200, and 400 μg/mL) demonstrate NDUFB5's protective effects

  • NDUFB5 overexpression counteracts AGEs-induced cellular damage

  • In vivo diabetic mouse models show improved wound healing with enhanced NDUFB5 expression

• Molecular pathway:

  • METTL3-mediated m6A modification increases NDUFB5 expression

  • This modification is recognized by IGF2BP2, enhancing mRNA stability

  • The METTL3-NDUFB5 axis represents a potential therapeutic target

• Methodological approaches:

  • Wound closure assays in diabetic mouse models

  • Laser Doppler imaging to assess wound perfusion

  • Histological assessment of vascularization and granulation tissue

  • Targeted manipulation of NDUFB5 expression in specific cell types

These findings suggest that enhancing NDUFB5 function could represent a novel therapeutic strategy for diabetic foot ulcers, a devastating complication with high morbidity .

What experimental models are suitable for studying NDUFB5 in the context of mitochondrial diseases?

For investigating NDUFB5's role in mitochondrial diseases, several experimental models offer complementary advantages:

• Cellular models:

  • Patient-derived fibroblasts carrying complex I deficiencies

  • CRISPR-engineered cell lines with NDUFB5 mutations or deletions

  • iPSC-derived neurons, cardiomyocytes, or myocytes for tissue-specific effects

  • Cybrid cells containing patient mitochondria in a controlled nuclear background

• Animal models:

  • Conditional tissue-specific Ndufb5 knockout mice

  • CRISPR-engineered animal models with specific pathogenic mutations

  • Diabetic mouse models for studying wound healing mechanisms

  • Drosophila models for high-throughput phenotypic screening

• Experimental analyses:

  • Comprehensive metabolic phenotyping

  • Tissue-specific respiratory chain function assessment

  • In vivo imaging of metabolic parameters

  • Lifespan and health span measurements

• Disease-specific considerations:

  • Models should recapitulate key features of the human condition

  • Developmental timing of NDUFB5 disruption may be critical

  • Consideration of compensatory mechanisms that may mask phenotypes

  • Combination with environmental stressors to reveal latent defects

When designing disease models, researchers should note that complete NDUFB5 deficiency might be embryonically lethal (as seen with other complex I subunits like Ndufa5 ), necessitating conditional approaches.

How can therapeutic targeting of the METTL3-NDUFB5 axis be assessed in experimental systems?

The METTL3-NDUFB5 regulatory axis represents a promising therapeutic target, particularly for conditions like diabetic foot ulcers . Methodological approaches for evaluating interventions include:

• Target validation strategies:

  • METTL3 overexpression rescues AGEs-induced cell injury via increased NDUFB5

  • Combined knockdown experiments (METTL3 expression with NDUFB5 siRNA) demonstrate pathway specificity

  • m6A site mutagenesis in NDUFB5 mRNA confirms direct regulation mechanism

• Therapeutic screening approaches:

  • Small molecule modulators of METTL3 activity

  • RNA-based therapies targeting NDUFB5 mRNA stability

  • Cell-based high-throughput screens using NDUFB5 expression reporters

  • Mitochondrial respiration as a functional readout

• Efficacy assessment techniques:

  • Cell viability and migration in AGEs-treated HUVECs

  • Mitochondrial respiration parameters via Seahorse analysis

  • In vivo wound healing in diabetic mouse models

  • Tissue-specific complex I activity measurements

• Analytical considerations:

  • Dose-response relationships for pathway modulators

  • Temporal dynamics of intervention effects

  • Off-target effects assessment via transcriptome/proteome analysis

  • Biomarker development for monitoring therapy response

The following experimental parameters have proven informative:

• AGEs treatment at 200 μg/ml for 48 hours in HUVECs

• METTL3 expression vector transfection with or without NDUFB5 siRNA

• Dual-luciferase assays with NDUFB5 3'UTR constructs

• Actinomycin D (0.2 mM) treatment to assess mRNA stability

How can structural biology techniques be applied to study Pan troglodytes NDUFB5 within complex I?

Structural analysis of Pan troglodytes NDUFB5 within complex I requires specialized approaches for membrane protein complexes:

• Cryo-electron microscopy (cryo-EM):

  • The method of choice for intact complex I structural determination

  • Sample preparation using detergent solubilization or nanodisc reconstitution

  • Classification approaches to identify conformational heterogeneity

  • Local refinement focusing on the NDUFB5 region

• Integrative structural biology:

  • Crosslinking mass spectrometry to identify distance constraints

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Electron paramagnetic resonance for specific distance measurements

  • Molecular dynamics simulations to model conformational changes

• Comparative analysis approaches:

  • Structural comparison between Pan troglodytes and human complex I

  • Mapping of conserved interfaces versus species-specific regions

  • Identification of potential binding pockets for small molecules

  • Structural basis for assembly checkpoint mechanisms

• Technical considerations:

  • Preparation of highly pure, homogeneous complex I samples

  • Stabilization strategies using amphipols or nanodiscs

  • Resolution limitations in specific regions of complex I

  • Validation of structural models using mutagenesis

The structural insights gained would complement functional studies and potentially reveal species-specific features of NDUFB5 within the complex I architecture.

What approaches can distinguish between NDUFB5's structural versus catalytic roles in complex I?

Distinguishing between structural and catalytic contributions of NDUFB5 requires sophisticated experimental designs:

• Structure-function mutagenesis:

  • Systematic alanine scanning mutagenesis of conserved residues

  • Charge reversal mutations at key interaction interfaces

  • Design of chimeric proteins swapping domains between species

  • Introduction of photocrosslinkable amino acids at specific positions

• Kinetic and thermodynamic analyses:

  • Steady-state kinetics of complex I with modified NDUFB5

  • Stopped-flow spectroscopy for pre-steady-state measurements

  • Thermal stability assessments of complex I with NDUFB5 variants

  • Assembly rate measurements with tagged subunits

• Computational approaches:

  • Molecular dynamics simulations of electron transfer pathways

  • Electrostatic surface mapping to identify potential functional sites

  • Normal mode analysis to identify dynamic communication pathways

  • Evolutionary covariance analysis to identify functionally coupled residues

• Specialized assays:

  • Site-specific electron transfer measurements

  • Proton pumping efficiency assessments

  • ROS production quantification with NDUFB5 variants

  • Complex I conformational change measurements

These complementary approaches can reveal whether NDUFB5 primarily contributes to complex I stability, regulates electron transfer, modulates proton pumping, or serves as an assembly factor.

How can evolutionary analysis of NDUFB5 inform our understanding of mitochondrial complex I function?

Evolutionary analysis of NDUFB5 provides valuable insights into complex I function and adaptation:

• Phylogenetic approaches:

  • Construction of phylogenetic trees based on NDUFB5 sequences

  • Comparison with trees based on core subunits

  • Analysis of evolutionary rates across different taxonomic groups

  • Identification of lineage-specific adaptations

• Selection pressure analysis:

  • Calculation of dN/dS ratios to identify selection signatures

  • Site-specific selection analysis to pinpoint functionally important residues

  • Comparison of selection patterns between primates and other mammals

  • Correlation with ecological or physiological adaptations

• Coevolution mapping:

  • Identification of coevolving residues within NDUFB5

  • Detection of coevolution between NDUFB5 and other complex I subunits

  • Mapping coevolving networks onto structural models

  • Experimental validation of predicted functional interactions

• Comparative functional analysis:

  • Cross-species complementation studies with NDUFB5 orthologs

  • Biochemical characterization of NDUFB5 from diverse lineages

  • Testing adaptation hypotheses through directed mutagenesis

  • Reconstruction of ancestral NDUFB5 sequences for functional testing

The high conservation observed across species (cow: 93%, mouse: 86%, zebrafish: 83%) suggests fundamental importance, while species-specific variations may reveal adaptive mechanisms that could inform therapeutic approaches for mitochondrial disorders.