Recombinant Vampyressa bidens NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its role in mitochondrial function?

MT-ND4L (NADH-ubiquinone oxidoreductase chain 4L) is a critical component of mitochondrial Complex I (NADH:ubiquinone oxidoreductase), which is the first and largest enzyme complex in the electron transport chain of oxidative phosphorylation. This protein is encoded by the mitochondrial genome rather than nuclear DNA. MT-ND4L functions as an integral membrane protein within the hydrophobic arm of Complex I that is embedded in the inner mitochondrial membrane . It contributes to the proton-pumping mechanism that helps establish the electrochemical gradient necessary for ATP synthesis. The complete amino acid sequence of Vampyressa bidens MT-ND4L consists of 98 amino acids: MSLTYMN MFMAFTISLLGLLMYRSHMMSSLLCLEGMLSLFVMMTMTILNTHTLASMIP IILLVFAACEAALGLSLLVMVSTTYGMDYVQNLNLLQC . This protein plays a crucial role in cellular energy production and mitochondrial function.

What is the evolutionary conservation pattern of MT-ND4L across species?

While the search results don't provide direct comparative data across multiple species, we can observe that MT-ND4L is highly conserved across mammalian species due to its essential role in energy metabolism. The Vampyressa bidens (Bidentate yellow-eared bat) MT-ND4L has structural features typical of mitochondrial membrane proteins, including hydrophobic transmembrane domains that anchor it within the inner mitochondrial membrane . The conservation of this protein reflects its critical role in oxidative phosphorylation, a fundamental process in aerobic energy production. Evolutionary conservation analysis would typically show higher preservation of functional domains involved in electron transport and proton pumping compared to regions with structural roles.

What are the optimal conditions for storing recombinant MT-ND4L protein?

Based on the product specifications for recombinant Vampyressa bidens MT-ND4L, the optimal storage conditions are as follows:

• Long-term storage: Store at -20°C or -80°C for extended periods

• Working aliquots: Store at 4°C for up to one week

• Storage buffer: Use a Tris-based buffer with 50% glycerol, optimized for protein stability

• Handling precautions: Avoid repeated freezing and thawing cycles, as this can lead to protein denaturation and loss of activity

These conditions help maintain the structural integrity and functional activity of the recombinant protein. The high glycerol content (50%) prevents ice crystal formation during freezing, which could otherwise damage the protein structure. Creating small working aliquots is recommended to minimize freeze-thaw cycles and preserve the remaining stock.

What techniques are most effective for studying MT-ND4L mutations and their functional impacts?

Multiple complementary techniques have proven effective for studying MT-ND4L mutations:

• PCR amplification: Specific primers can be designed to target the MT-ND4L gene region for initial identification of variants. For example, researchers have used the primer pair 5'-TCTGGCCTATGAGTGACTAC-3' (forward) and 5'-ACTGTGAGTGCGTTCGTTCGTAGTTTGAG-3' (reverse) with an annealing temperature of 54°C to generate a 1,415 bp PCR product covering the MT-ND4L region .

• Whole exome sequencing (WES): This technique has been successfully employed to identify mitochondrial variants embedded within nuclear DNA sequencing data, as demonstrated in the Alzheimer's Disease Sequencing Project which identified the rs28709356 C>T variant in MT-ND4L .

• SKAT-O gene-based testing: This statistical approach allows researchers to evaluate the collective impact of all variants within MT-ND4L rather than focusing on individual mutations .

• Functional prediction analysis: Computational tools can predict whether identified mutations are likely to be deleterious to protein stability and function. This approach has been used to analyze MT-ND4 mutations, determining that some variants (like m.11519A>C) have direct impacts on protein stability and complex I function .

How can researchers effectively express recombinant MT-ND4L for functional studies?

When expressing recombinant MT-ND4L, researchers should consider:

• Expression system selection: Due to MT-ND4L's hydrophobic nature and mitochondrial origin, specialized expression systems that can handle membrane proteins are preferred. Bacterial systems may require optimization with specialized strains designed for membrane protein expression.

• Codon optimization: Since MT-ND4L is encoded by mitochondrial DNA, its codon usage differs from nuclear genes. Codon optimization for the selected expression system improves protein yield.

• Fusion tags: The inclusion of appropriate tags (determined during the production process) facilitates purification while minimizing interference with protein function .

• Expression region: For Vampyressa bidens MT-ND4L, the full expression region covers amino acids 1-98 .

• Purification strategy: A balanced approach is needed to solubilize the membrane protein while maintaining its native conformation, typically using mild detergents during extraction and purification.

• Functional validation: Activity assays specific to NADH dehydrogenase (EC 1.6.5.3) function should be performed to confirm that the recombinant protein retains its enzymatic activity .

What is the association between MT-ND4L variants and Alzheimer's disease?

Research from the Alzheimer's Disease Sequencing Project has identified a significant association between MT-ND4L variants and Alzheimer's disease (AD). Analysis of 4,220 mtDNA variants from 10,831 participants revealed a study-wide significant association of AD with a rare MT-ND4L variant (rs28709356 C>T), which has a minor allele frequency of 0.002 and a P-value of 7.3 × 10⁻⁵ . Furthermore, gene-based testing demonstrated a significant association between MT-ND4L as a whole and AD risk (P = 6.71 × 10⁻⁵) .

This association suggests that mitochondrial dysfunction, particularly related to Complex I activity, may play a role in AD pathogenesis. Since MT-ND4L is a crucial component of Complex I, variants that affect its function could impair energy metabolism in neurons, potentially contributing to neurodegeneration. The high energy demands of neural tissue make it particularly vulnerable to mitochondrial dysfunction, which may explain the association between MT-ND4L variants and neurodegenerative diseases like AD.

How do mutations in MT-ND4L affect Complex I assembly and function?

Mutations in MT-ND4L can disrupt Complex I assembly and function through several mechanisms:

• Disruption of protein stability: Deleterious mutations can destabilize the MT-ND4L protein structure, affecting its integration into the membrane arm of Complex I. For example, mutations like those identified in related ND subunits (m.11519A>C, m.11523A>C) can have direct impacts on protein stability .

• Impaired complex assembly: Since Complex I assembly occurs through the formation and joining of discrete intermediates, mutations in MT-ND4L can prevent proper formation of the membrane arm intermediates. Research in Neurospora crassa has shown that the membrane arm forms through the association of a smaller 200 kDa and a larger 350 kDa assembly intermediate . Disruption of this process can lead to incomplete Complex I assembly.

• Cumulative effects of multiple mutations: Multiple mutations within the same gene can have cumulative effects on protein function. For instance, three mutations observed in the related ND4 gene (m.11519A>C, m.11523A>C, and m.11527C>T) were predicted to cause a cumulative destabilizing effect on the protein, potentially disrupting Complex I function .

• Reduced enzymatic activity: Mutations can directly affect the catalytic function of Complex I (NADH dehydrogenase, EC 1.6.5.3), reducing electron transfer efficiency and proton pumping capacity .

What is the potential role of MT-ND4L dysfunction in multiple sclerosis pathology?

While the search results specifically discuss mutations in the related ND4 gene rather than ND4L in multiple sclerosis (MS), the findings have relevant implications for understanding how mitochondrial complex I dysfunction might contribute to MS pathology. Research has identified novel mutations in the mtDNA-encoded ND4 gene in patients with MS that could lead to complex I dysfunction . Since ND4 and ND4L are both critical components of mitochondrial complex I, dysfunction in either could potentially contribute to MS pathology through similar mechanisms.

The potential mechanisms by which MT-ND4L dysfunction might contribute to MS include:

• Energy deficiency: Neurons have high energy demands, and impaired complex I function could lead to insufficient ATP production, particularly affecting the energy-intensive saltatory conduction in myelinated axons.

• Increased reactive oxygen species (ROS) production: Dysfunctional complex I can increase ROS generation, contributing to oxidative stress and damage to myelin and axons.

• Compromised mitochondrial membrane potential: MT-ND4L is involved in proton pumping across the inner mitochondrial membrane; dysfunction could disrupt the electrochemical gradient necessary for ATP synthesis.

• Potential autoimmune triggering: Mitochondrial dysfunction could release mitochondrial components that act as damage-associated molecular patterns (DAMPs), potentially triggering or exacerbating autoimmune responses.

How can MT-ND4L be used as a model for studying mitochondrial complex I assembly?

MT-ND4L serves as an excellent model for studying mitochondrial complex I assembly for several reasons:

• Strategic position within complex I: MT-ND4L is located in the membrane arm of complex I, which forms through the association of discrete assembly intermediates . By tracking the incorporation of MT-ND4L, researchers can monitor specific stages of complex I assembly.

• Chaperone-dependent assembly: Research has identified novel chaperones specifically involved in complex I membrane arm assembly . MT-ND4L incorporation depends on these chaperones, making it a useful marker for studying chaperone-dependent assembly mechanisms.

• Cross-species conservation: Despite some sequence variations, the fundamental role of MT-ND4L in complex I is conserved across species, allowing for comparative studies between model organisms like Neurospora crassa and mammals .

• Disease-relevant mutations: The identification of disease-associated mutations in MT-ND4L, such as the AD-associated rs28709356 C>T variant, provides natural experiments for understanding how specific protein alterations affect assembly and function .

Researchers can use pulse-chase labeling experiments to track MT-ND4L incorporation into assembly intermediates, similar to studies that have shown how specific chaperones are repeatedly involved in many assembly cycles of the membrane arm intermediate .

What are the implications of MT-ND4L research for developing mitochondrial-targeted therapeutics?

Research on MT-ND4L has several implications for developing mitochondrial-targeted therapeutics:

• Biomarker identification: The association between MT-ND4L variants and diseases like Alzheimer's suggests that specific mitochondrial genotypes might serve as biomarkers for disease risk or progression . These biomarkers could help identify patients who might benefit from mitochondrial-targeted interventions.

• Target validation: Understanding how specific MT-ND4L mutations affect complex I function provides validated targets for therapeutic intervention. For example, the knowledge that certain mutations destabilize the protein suggests that therapies designed to stabilize mutant MT-ND4L might be effective.

• Bypass strategies: For cases where MT-ND4L dysfunction leads to complex I deficiency, developing compounds that can bypass complex I and deliver electrons directly to later components of the respiratory chain could be therapeutic.

• Mitochondrial replacement therapy: In severe cases of MT-ND4L mutation-related disease, mitochondrial replacement therapy could potentially prevent transmission of pathogenic mitochondrial DNA mutations.

• Chaperone modulation: The identification of specific chaperones involved in complex I assembly suggests that therapies enhancing chaperone function could potentially improve the assembly of complexes containing mutant MT-ND4L.

What comparative analyses can be performed between MT-ND4L from Vampyressa bidens and other mammalian species?

Researchers can conduct several valuable comparative analyses between Vampyressa bidens MT-ND4L and its counterparts in other mammalian species:

• Sequence homology analysis: Comparing the amino acid sequence of Vampyressa bidens MT-ND4L (MSLTYMN MFMAFTISLLGLLMYRSHMMSSLLCLEGMLSLFVMMTMTILNTHTLASMIP IILLVFAACEAALGLSLLVMVSTTYGMDYVQNLNLLQC) with sequences from other mammals can identify conserved regions likely crucial for function versus variable regions that might reflect species-specific adaptations.

• Structural modeling comparisons: Creating 3D models of MT-ND4L from different species can reveal how structural variations might affect protein stability, membrane integration, or interactions with other complex I subunits.

• Evolutionary rate analysis: Calculating the rate of evolutionary change in different regions of MT-ND4L across species can identify functional constraints and potential regions under positive selection.

• Disease-associated variant mapping: Mapping known disease-associated variants (like the AD-associated rs28709356 C>T) across species can help determine whether the same positions are conserved or variable, providing insight into their functional importance.

• Bat-specific adaptations: As Vampyressa bidens is a bat species, comparative analysis could reveal adaptations related to the unique metabolic demands of flight or the bat's daily torpor cycles, which involve temperature fluctuations that might affect mitochondrial protein function .

What is the most effective experimental design for studying MT-ND4L mutations in cellular models?

An effective experimental design for studying MT-ND4L mutations should include:

• CRISPR/Cas9 mitochondrial genome editing: While challenging due to the mitochondrial location, newer techniques allow for targeted mitochondrial DNA editing to introduce specific MT-ND4L mutations of interest.

• Cybrid cell technology: Creating transmitochondrial cybrids by fusing cells depleted of mitochondrial DNA with platelets or enucleated cells containing mitochondria with the MT-ND4L mutation of interest. This approach maintains a consistent nuclear background while allowing comparison of different mitochondrial genotypes.

• Control selection: Isogenic controls should be used, differing only in the MT-ND4L mutation status. This might include:

  • Wild-type cells

  • Cells with synonymous MT-ND4L mutations (not affecting protein sequence)

  • Cells with mutations corrected back to wild-type sequence

• Functional assays:

  • Complex I activity measurements (NADH:ubiquinone oxidoreductase activity, EC 1.6.5.3)

  • Oxygen consumption rate determination

  • ATP production quantification

  • Reactive oxygen species measurement

  • Mitochondrial membrane potential assessment

• Assembly analysis:

  • Blue native PAGE to assess complex I assembly

  • Immunoprecipitation to study interaction with assembly factors

  • Pulse-chase labeling to track incorporation into complex I, similar to techniques used in Neurospora crassa studies

• Cell stress challenges:

  • Assess how MT-ND4L mutations affect cellular response to metabolic stress

  • Test response to oxidative stress, reflecting conditions in neurodegenerative diseases

What analytical techniques are most appropriate for characterizing recombinant MT-ND4L protein?

For comprehensive characterization of recombinant MT-ND4L protein, researchers should employ multiple complementary analytical techniques:

• Primary structure confirmation:

  • Mass spectrometry to verify the exact molecular weight and confirm the amino acid sequence

  • N-terminal sequencing to confirm the starting amino acid (Methionine in Vampyressa bidens MT-ND4L)

  • Peptide mapping to verify complete sequence coverage

• Secondary and tertiary structure analysis:

  • Circular dichroism spectroscopy to assess secondary structure components (alpha helices, beta sheets)

  • Fourier-transform infrared spectroscopy for membrane proteins

  • Limited proteolysis to identify accessible regions versus protected domains

• Functional characterization:

  • NADH:ubiquinone oxidoreductase activity assays (EC 1.6.5.3)

  • Electron paramagnetic resonance to study electron transfer properties

  • Proton pumping assays in reconstituted proteoliposomes

• Interaction studies:

  • Surface plasmon resonance to study interactions with other complex I subunits

  • Cross-linking studies to identify neighboring proteins in assembled complex

  • Blue native PAGE to assess incorporation into complex I

• Stability assessment:

  • Differential scanning calorimetry to determine thermal stability

  • Storage stability testing under different conditions (as recommended: -20°C or -80°C for long-term, 4°C for working aliquots)

  • Freeze-thaw stability assessment

• Structural biology approaches:

  • Cryo-electron microscopy as part of reconstituted complex I

  • X-ray crystallography (challenging for membrane proteins but potentially informative)

  • NMR spectroscopy for specific domains or in membrane-mimetic environments

How can contradictory findings in MT-ND4L research be reconciled through experimental design?

Contradictory findings in MT-ND4L research can be reconciled through strategic experimental design approaches:

• Standardized methodologies: Establish consistent protocols for:

  • MT-ND4L isolation and purification

  • Complex I activity measurement

  • MT-ND4L mutation detection and characterization

  • Data normalization and statistical analysis

• Cross-validation with multiple techniques: When contradictory findings exist regarding MT-ND4L function or disease association, employ multiple independent methodologies to test the same hypothesis. For example, if contradictory findings exist about the AD-associated rs28709356 C>T variant , validate using:

  • Different cohorts and populations

  • Multiple genetic analysis methods

  • Functional studies in cellular models

  • In vivo model systems

• Comprehensive mutation analysis: Study the effects of mutations in isolation and in combination, as multiple mutations can have cumulative effects on protein function. For example, studies of the related ND4 gene found that three mutations (m.11519A>C, m.11523A>C, and m.11527C>T) together caused a cumulative destabilizing effect .

• Nuclear-mitochondrial interactions: Account for the influence of nuclear genetic background on MT-ND4L phenotypes, as nuclear-encoded proteins interact with MT-ND4L in complex I assembly and function .

• Meta-analysis approach: When conflicting results appear in the literature, conduct systematic reviews and meta-analyses to:

  • Identify patterns across studies

  • Assess the quality of contradictory evidence

  • Determine whether differences might be explained by methodological variations

  • Calculate cumulative effect sizes across studies

• Collaborative multi-center studies: Establish research consortia to:

  • Share standardized protocols

  • Analyze identical samples across multiple laboratories

  • Pool data for increased statistical power

  • Cross-validate findings in diverse populations

This comprehensive approach can help reconcile contradictory findings and establish a more consistent understanding of MT-ND4L function and its role in diseases like Alzheimer's and potentially multiple sclerosis .

Data Table: Comparison of MT-ND4L Characteristics and Research Applications