Recombinant Mustela vison NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its structural composition in Mustela vison?

MT-ND4L is a gene of the mitochondrial genome coding for the NADH-ubiquinone oxidoreductase chain 4L (ND4L) protein. In Mustela vison (American mink), the full-length protein consists of 98 amino acids with the sequence: MSMVYINIFLAFTLSFMGLLIYRSSHLMSSLLCLEGMMLSLFVMMTITILINHTLASMTPIILLVFAACEAALGLSLLVMISTTYGTDYVQNLNLLQC . The protein has a molecular weight of approximately 11 kDa and represents one of the most hydrophobic subunits of Complex I in the respiratory chain . The hydrophobic nature of this protein contributes to its localization within the transmembrane domain of the complex.

What is the function of MT-ND4L in mitochondrial energy production?

MT-ND4L protein functions as a critical subunit of NADH dehydrogenase (ubiquinone), also known as Complex I of the electron transport chain . This complex is located in the mitochondrial inner membrane and is the largest of the five complexes involved in oxidative phosphorylation. Methodologically, the function of MT-ND4L can be assessed through:

• Complex I activity assays measuring NADH:ubiquinone oxidoreductase activity

• Oxygen consumption measurements in isolated mitochondria

• ATP production assays in cellular systems

The protein participates in the first step of the electron transport process, facilitating the transfer of electrons from NADH to ubiquinone, which creates an electrochemical gradient across the inner mitochondrial membrane that drives ATP synthesis .

How should recombinant MT-ND4L be properly stored and handled in laboratory settings?

For optimal stability and activity of recombinant Mustela vison MT-ND4L:

• Store the lyophilized protein at -20°C to -80°C upon receipt

• After reconstitution, add 5-50% glycerol (with 50% being optimal for long-term storage)

• Aliquot to avoid repeated freeze-thaw cycles

• For working solutions, store at 4°C for up to one week

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

Storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0, although this may vary between preparations .

What are the most effective expression systems for producing functional recombinant MT-ND4L?

For hydrophobic mitochondrial proteins like MT-ND4L, addition of solubilization tags (His, GST, MBP) and optimization of detergent conditions during purification are critical for obtaining functional protein .

How can researchers effectively study the interaction between MT-ND4L and other Complex I subunits?

To study subunit interactions within Complex I:

• Co-immunoprecipitation assays using antibodies against MT-ND4L or other subunits

• Crosslinking coupled with mass spectrometry to identify interaction sites

• Blue Native PAGE to analyze intact Complex I assembly

• Proximity labeling techniques (BioID or APEX) to identify proteins in close proximity to MT-ND4L

• Cryo-electron microscopy for structural analysis of the entire complex

These methods help reveal how MT-ND4L, as one of the most hydrophobic subunits, contributes to the core structure of the transmembrane region of Complex I .

What is the evidence linking MT-ND4L mutations to Leber's Hereditary Optic Neuropathy (LHON)?

Mutations in the MT-ND4L gene have been identified in several families with Leber's Hereditary Optic Neuropathy (LHON) . A specific mutation, T10663C (Val65Ala), has been documented to change a single amino acid in the protein, replacing valine with alanine at position 65 . Research approaches to study this association include:

• Family-based genetic studies tracking mutation inheritance patterns

• Functional analysis of mutant proteins in cellular and animal models

• Biochemical assessment of Complex I activity in patient-derived samples

• Measurement of reactive oxygen species production in cells harboring mutations

• Evaluation of mitochondrial membrane potential and ATP synthesis rates

While the exact pathophysiological mechanism remains under investigation, disruption of Complex I function appears to particularly affect retinal ganglion cells, resulting in the characteristic vision loss in LHON .

How does MT-ND4L contribute to metabolic disorders and what research models are available?

MT-ND4L dysfunction has been associated with metabolic disorders including:

• Increased BMI and obesity in adults

• Altered metabolite profiles, particularly involving phosphatidylcholine ratios

• Potential links to type 2 diabetes

Research has shown that the variant mt10689 G>A located in the MT-ND4L gene is associated with 16 different metabolite ratios, all involving phosphatidylcholine diacyl C36:6 (PC aa C36:6) . These associations may explain pathways involved in the development of metabolic conditions, as PC aa C36:6 has been associated with patterns of fat concentration in the body, including visceral fat and liver fat content .

Research models for studying these associations include:

• Cellular models with engineered MT-ND4L variants

• Metabolomic profiling of patient cohorts

• Transgenic animal models with altered MT-ND4L expression

• Population-based association studies like the KORA-F4 study

What is the significance of MT-ND4L variants in Alzheimer's disease research?

Recent research has identified a rare MT-ND4L variant (rs28709356 C>T; minor allele frequency = 0.002) with significant association to Alzheimer's disease (AD) risk (P = 7.3 × 10⁻⁵) . This finding from the Alzheimer's Disease Sequencing Project (ADSP), which analyzed 10,831 participants, provides compelling evidence for mitochondrial involvement in AD pathogenesis .

Research methodologies to investigate this association include:

• Whole exome sequencing with specialized pipelines for accurate assembly and variant calling in mitochondrial genomes

• Association testing using statistical methods like the SCORE test for individual variants and SKAT-O for gene-based tests

• Expression analysis comparing MT-ND4L levels between AD cases, mild cognitive impairment cases, and controls

• Functional studies of how the variant affects Complex I activity and mitochondrial function in neuronal models

The significant findings in MT-ND4L provide evidence for mitochondrial dysfunction as a potential contributor to AD development .

How can researchers investigate the unusual gene overlap between MT-ND4L and MT-ND4?

The human MT-ND4L gene has a unique 7-nucleotide gene overlap with MT-ND4, where the last three codons of MT-ND4L (5'-CAA TGC TAA-3' coding for Gln, Cys and Stop) overlap with the first three codons of MT-ND4 (5'-ATG CTA AAA-3' coding for Met-Leu-Lys) . To investigate this unusual genetic feature:

• Employ ribosome profiling to analyze translation efficiency at the overlapping region

• Use CRISPR-based approaches with precision edits to study the functional importance of this overlap

• Conduct comparative genomics analyses across species to determine evolutionary conservation

• Perform in vitro translation studies with various constructs to assess the impact on protein expression

• Utilize reporter gene assays to measure expression efficiency

This overlapping gene structure represents an interesting case of genetic economy in the mitochondrial genome and may have implications for coordinated expression of these related subunits .

What methodologies are most effective for studying heteroplasmy in MT-ND4L mutations?

Heteroplasmy—the presence of multiple mitochondrial DNA variants within a single cell—presents unique challenges for MT-ND4L research. Effective methodologies include:

• Next-Generation Sequencing (NGS) with high coverage (>3500-fold is recommended) to accurately detect low-level heteroplasmic variants

• Long-range PCR approaches to specifically amplify the mitochondrial genome prior to sequencing

• Digital droplet PCR for precise quantification of heteroplasmy percentages

• Single-cell sequencing to evaluate heteroplasmy distribution across different cells

• Pyrosequencing for targeted analysis of specific known variants

Researchers should be aware that deep sequencing is necessary for reliable detection of low-level heteroplasmy, as conventional methods may miss variants present at less than 15% frequency .

How do metabolomic profiles correlate with MT-ND4L variants, and what analytical approaches are recommended?

Studies have identified significant associations between MT-ND4L variants and metabolite profiles, particularly:

• The variant mt10689 G>A in MT-ND4L is associated with 16 different metabolite ratios, all involving phosphatidylcholine diacyl C36:6 (PC aa C36:6)

• These associations suggest MT-ND4L may influence lipid metabolism, particularly phosphatidylcholines which are critical for cell membrane structure and function

Recommended analytical approaches include:

For optimal results, researchers should consider both log-transformed and untransformed metabolite ratios, as different transformations may affect the detection of associations .

How does MT-ND4L structure and function compare between Mustela vison and other mammalian species?

Comparative analysis of MT-ND4L across mammalian species reveals both conservation and variation:

Research methods for comparative studies include:

• Multiple sequence alignment to identify conserved residues and domains

• Homology modeling based on known structures

• Evolutionary rate analysis to identify regions under selective pressure

• Functional complementation studies in model systems

• Phylogenetic analysis to trace the evolutionary history of the gene

The high degree of conservation in key functional domains suggests essential roles in Complex I function across diverse mammalian lineages .

What experimental approaches can determine if findings from MT-ND4L studies in one species can be extrapolated to others?

To evaluate the transferability of MT-ND4L research findings across species:

• Heterologous expression studies: Express MT-ND4L from different species in the same cellular background to compare functional properties

• Cross-species complementation: Test if MT-ND4L from one species can rescue phenotypes in cells with deficient MT-ND4L from another species

• Conservation analysis: Determine the evolutionary conservation of specific residues implicated in disease or function

• Site-directed mutagenesis: Introduce equivalent mutations across species to compare effects

• Enzymatic assays: Compare biochemical properties of recombinant proteins from different species

These approaches are particularly important when using model organisms or recombinant proteins as proxies for human mitochondrial diseases associated with MT-ND4L mutations .

What are the common challenges in expressing and purifying recombinant MT-ND4L and how can they be addressed?

MT-ND4L poses significant technical challenges due to its hydrophobic nature and membrane localization:

For recombinant preparations, maintaining protein in detergent micelles or reconstituting into liposomes/nanodiscs is often necessary to preserve native-like structure and function .

How can researchers verify the functional integrity of recombinant MT-ND4L after purification?

To ensure that purified recombinant MT-ND4L retains its structural and functional properties:

• Structural integrity assessment:

  • Circular dichroism spectroscopy to confirm secondary structure content

  • Limited proteolysis to verify proper folding

  • Size exclusion chromatography to evaluate oligomeric state

  • Thermal shift assays to measure protein stability

• Functional validation:

  • NADH:ubiquinone oxidoreductase activity assays

  • Reconstitution into proteoliposomes for membrane potential measurements

  • Binding assays with known interaction partners

  • Electron paramagnetic resonance to assess redox center integrity

• Complex I assembly assessment:

  • Co-reconstitution with other subunits to test complex formation

  • Blue Native PAGE to verify incorporation into larger complexes

  • Cryo-EM to visualize protein structure within the complex

Researchers should select validation methods appropriate for their specific research questions while considering that fully functional Complex I typically requires multiple subunits beyond MT-ND4L alone .