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

What is the structural organization of gorilla NDUFB5 and how does it compare to human NDUFB5?

Gorilla NDUFB5, like its human counterpart, has a distinctive two-domain structure with an N-terminal hydrophobic domain and a C-terminal hydrophilic domain. The protein weighs approximately 21.7 kDa and consists of 189 amino acids in humans . The N-terminal hydrophobic domain forms an alpha helix spanning the inner mitochondrial membrane, while the C-terminal hydrophilic domain interacts with globular subunits of Complex I .

Methodological approach: To analyze structural differences between gorilla and human NDUFB5, researchers should:

• Perform sequence alignment using tools like CLUSTAL W

• Generate 3D protein models using homology modeling

• Compare conserved domains and motifs using predictive algorithms

• Validate structural differences using X-ray crystallography or cryo-EM

How does gorilla NDUFB5 contribute to mitochondrial respiratory function?

While NDUFB5 is not directly involved in the catalytic activity of Complex I, it plays a critical role in maintaining mitochondrial respiration. Research indicates that NDUFB5 acts as an anchor for the NADH dehydrogenase complex at the inner mitochondrial membrane . In diabetic models, upregulation of NDUFB5 accelerates wound healing by improving mitochondrial respiratory function .

To study NDUFB5's contribution to respiratory function:

• Use oxygen consumption rate (OCR) measurements in isolated mitochondria

• Examine electron transport chain complex assembly using blue native gel electrophoresis

• Assess complex I activity using NADH:ubiquinone oxidoreductase assays in models with NDUFB5 knockdown/overexpression

How has NDUFB5 evolved across primates, and what can this tell us about its functional importance?

Evolutionary analysis reveals that NDUFB5 shows variable patterns of sequence conservation across primates. Studies analyzing mitochondrial proteins in humans, chimpanzees, and gorillas found that NADH dehydrogenase subunits exhibited distinct evolutionary patterns . This suggests selective pressures acting on respiratory chain components.

Research methodology:

• Perform phylogenetic analysis using maximum likelihood or Bayesian methods

• Calculate sequence divergence using restriction enzyme cut sites analysis

• Analyze synonymous vs. non-synonymous substitution rates (dN/dS ratio)

• Examine patterns of selection using tests like McDonald-Kreitman

Table 1. Sequence divergence patterns observed in mitochondrial genes across primate species:

Note: Data adapted from evolutionary analysis studies

How can researchers distinguish between nuclear insertions of mitochondrial DNA (numts) and authentic mitochondrial NDUFB5 in gorilla genetic studies?

Nuclear insertions of mitochondrial DNA (numts) pose significant challenges in evolutionary studies of mitochondrial genes like NDUFB5 in gorillas. These nuclear copies can confound genetic analyses and lead to inaccurate evolutionary interpretations .

Methodological solution:

• Enrich samples for mitochondrial DNA using differential centrifugation

• Design PCR primers specific to conserved regions flanking the gene of interest

• Implement an anchored-PCR strategy to bias amplification toward nuclear copies:

  • Isolate nuclear-enriched DNA using fibroblast cell cultures

  • Use long-range PCR with high-fidelity polymerase (e.g., TaKaRa LA Taq)

  • Clone PCR products and sequence multiple clones to identify variant forms

• Compare sequences with known mitochondrial reference genomes

• Validate findings using BAC genomic libraries to identify and characterize numts

What are the most effective methods for expressing recombinant gorilla NDUFB5 for structural and functional studies?

For successful expression of gorilla NDUFB5:

• Vector selection and design:

  • Use the pcDNA3.1(+) plasmid system for mammalian expression

  • For bacterial expression, consider pET systems with solubility tags (MBP, SUMO, or GST)

  • Include appropriate targeting sequences for mitochondrial localization

• Expression systems:

  • HUVECs have been successfully used for NDUFB5 expression studies

  • For higher yields, consider HEK293 cells with tetracycline-inducible promoters

  • For crystallography studies, insect cell systems (Sf9/Sf21) may provide better protein folding

• Transfection methods:

  • Use Lipofectamine 2000 following established protocols for mammalian cells

  • Optimize transfection conditions: DNA:lipid ratio (1:2.5-1:3), cell density (70-80% confluence)

  • For stable cell lines, select with appropriate antibiotics for 2-3 weeks

What purification strategies yield the highest purity and activity for recombinant gorilla NDUFB5?

Given NDUFB5's hydrophobic nature and membrane association, specialized purification approaches are required:

• Cell lysis and membrane fraction isolation:

  • Use mild detergents (0.5-1% DDM or 1% digitonin) to solubilize membrane proteins

  • Differential centrifugation (10,000×g for mitochondrial fraction, 100,000×g for membrane fraction)

  • Sonication in the presence of protease inhibitors

• Column chromatography sequence:

  • Ni-NTA affinity chromatography for His-tagged proteins

  • Ion exchange chromatography using salt gradient elution

  • Size exclusion chromatography for final polishing

  • Consider hydroxyapatite chromatography for membrane proteins

• Activity preservation:

  • Maintain samples at 4°C throughout purification

  • Include 10-15% glycerol in all buffers

  • Avoid freeze-thaw cycles; store aliquots at -80°C

How does m6A modification affect NDUFB5 expression and function in mitochondrial biology?

Recent research has revealed that METTL3-mediated m6A modification plays a crucial role in regulating NDUFB5 expression . This epigenetic regulation has significant implications for mitochondrial function and cellular responses to stress.

Experimental approach to study m6A modification of NDUFB5:

• Identify m6A modification sites:

  • Perform RNA immunoprecipitation (RIP) using anti-m6A antibodies

  • Analyze through qRT-PCR to determine enrichment of m6A in NDUFB5 3'UTR

  • Validate using luciferase reporter assays with NDUFB5 3'UTR

• Measure modification effects on expression:

  • Assess mRNA stability using actinomycin D treatment (0.2 mM) at time intervals (0, 2, 4, 6h)

  • Quantify NDUFB5 mRNA levels via qRT-PCR using primers:

    • NDUFB5-F: 5ʹ-TCCTGTTCGACACAGTGGAG-3ʹ

    • NDUFB5-R: 5ʹ-AGGACGGCCATTGTTCTTTCA-3ʹ

• Functional consequences:

  • Overexpress or knock down METTL3 using expression vectors or siRNAs

  • Measure cell viability, migration, and mitochondrial respiration

  • Examine protein interaction networks using co-immunoprecipitation

Research findings indicate that METTL3-mediated NDUFB5 m6A modification promotes cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, suggesting potential therapeutic applications for conditions like diabetic foot ulcers .

How can gorilla NDUFB5 studies inform our understanding of mitochondrial dysfunction in human neurological disorders?

Mitochondrial dysfunction is implicated in numerous neurological disorders, and comparative studies of NDUFB5 across primates can provide valuable insights:

• Comparative expression analysis:

  • In Alzheimer's disease (AD) models, multiple NDUF subunits show reduced expression, including NDUFB5 (posterior error probability = 2.4e-2)

  • Analyze expression patterns in different brain regions and developmental stages

• Disease model applications:

  • Generate transgenic models expressing gorilla vs. human NDUFB5

  • Assess impact on mitochondrial function, oxidative stress, and neuronal health

  • Examine protein-protein interactions within Complex I

• Therapeutic implications:

  • Test whether gorilla NDUFB5 variants show differential resistance to oxidative stress

  • Develop peptide mimetics based on uniquely conserved regions

  • Evaluate potential for gene therapy approaches

What are the key technical challenges in analyzing NDUFB5 incorporation into Complex I and how can they be overcome?

Studying NDUFB5 incorporation into Complex I presents several technical challenges:

• Complex I assembly analysis:

  • Use blue native gel electrophoresis to assess complex formation

  • Knockout cell lines show limited assembly of the N-module, particularly affecting core subunits NDUFS1, NDUFV1, and NDUFV2

  • Implement in-gel activity assays to assess functional complex assembly

• Interaction mapping:

  • Perform crosslinking mass spectrometry to identify interaction sites

  • Consider proximity labeling approaches (BioID, APEX) for in vivo interaction mapping

  • Use cryo-EM for structural determination of the entire Complex I

• Functional assessment:

  • Implement Seahorse XF analysis for measuring oxygen consumption rate

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

  • Measure superoxide production using MitoSOX

How can researchers assess whether NDUFB5 mutations are pathogenic versus neutral evolutionary variants?

Distinguishing pathogenic mutations from neutral variants requires multiple lines of evidence:

• Evolutionary constraint analysis:

  • Studies of mitochondrial DNA variation in humans and chimpanzees show that the ratio of replacement to silent mutations is higher within species than between species

  • This pattern suggests many mitochondrial protein polymorphisms are slightly deleterious

• Functional validation approaches:

  • Express variants in knockout cell lines to test for functional rescue

  • Assess mitochondrial morphology and function

  • Measure complex I activity, ATP production, and reactive oxygen species generation

• Clinical correlation:

  • Compare sequence variations with known pathogenic mutations in human NDUFB5 homologs

  • Assess conservation at sites of variation across species

  • Evaluate structural impacts using protein modeling

Table 2. Assessment criteria for NDUFB5 variant pathogenicity:

What emerging technologies could revolutionize gorilla NDUFB5 research?

Several cutting-edge technologies hold promise for advancing NDUFB5 research:

• Cryo-electron microscopy:

  • Single-particle analysis for high-resolution structures

  • In situ structural determination within mitochondrial membranes

  • Time-resolved cryo-EM to capture conformational changes during electron transport

• CRISPR-based approaches:

  • Prime editing for precise modification of NDUFB5 sequences

  • CRISPR interference/activation for temporal control of expression

  • Base editing for introducing specific mutations modeling gorilla variants

• Single-cell technologies:

  • Single-cell RNA-seq to assess cell-specific expression patterns

  • Spatial transcriptomics to map NDUFB5 expression in tissue contexts

  • Single-cell proteomics to analyze NDUFB5 protein levels and modifications

• Organoid models:

  • Brain organoids for studying NDUFB5 function in a human neural context

  • Mitochondrial reporter systems for live imaging of function

  • Patient-derived organoids incorporating gorilla NDUFB5 variants

By leveraging these technologies and methodological approaches, researchers can gain deeper insights into the structure, function, and evolution of gorilla NDUFB5, potentially unlocking new therapeutic strategies for mitochondrial disorders.