Recombinant Tetraodon nigroviridis NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

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

MT-ND4L is a gene in the mitochondrial genome coding for the NADH-ubiquinone oxidoreductase chain 4L (ND4L) protein. This protein functions as a subunit of NADH dehydrogenase (ubiquinone), also known as Complex I, which is located in the mitochondrial inner membrane and represents the largest of the five complexes in the electron transport chain . The protein enables NADH dehydrogenase activity and is involved in mitochondrial electron transport from NADH to ubiquinone and proton motive force-driven mitochondrial ATP synthesis . MT-ND4L and other mitochondrially encoded subunits are highly hydrophobic and form the core of the transmembrane region of Complex I, which is essential for cellular energy production .

How is the MT-ND4L gene structured in Tetraodon nigroviridis compared to humans?

In humans, the MT-ND4L gene is located in mitochondrial DNA from base pair 10,469 to 10,765, producing an 11 kDa protein composed of 98 amino acids . While the search results don't provide specific details about the gene structure in Tetraodon nigroviridis, mitochondrial genes are generally conserved across species with variations that can provide insights into evolutionary relationships. An unusual feature of the human MT-ND4L gene is its 7-nucleotide gene overlap with the first three codons of the MT-ND4 gene, creating a reading frame shift . Research on Tetraodontiform fishes (which include Tetraodon nigroviridis) has shown that MT-ND4L, like other mitochondrial genes, exhibits evolutionary selection pressure that can vary across different lineages, suggesting functional importance in adaptation .

What experimental systems are used to study recombinant MT-ND4L protein function?

For studying recombinant MT-ND4L protein function, researchers typically employ various expression systems including bacterial (E. coli), yeast, insect cell, and mammalian cell systems. Each system offers different advantages for studying the hydrophobic membrane proteins like MT-ND4L. Recombinant expression allows for controlled studies of protein-protein interactions, enzymatic activity, and structural analyses. For Tetraodon nigroviridis MT-ND4L specifically, recombinant proteins can be produced and purified for use in enzyme-linked immunosorbent assays (ELISA) and other biochemical assays to evaluate its functional properties . When working with this highly hydrophobic protein, detergent solubilization or membrane mimetic systems such as nanodiscs or liposomes are often necessary to maintain native-like function during in vitro studies.

How do mutations in MT-ND4L contribute to disease phenotypes across species?

Mutations in MT-ND4L have been implicated in several human diseases, most notably Leber's Hereditary Optic Neuropathy (LHON) . A study of an Arab family from Kuwait with 14 affected male members identified two concurrent mutations in the ND4L gene (10609T>C and 10663T>C) that led to non-conservative amino acid changes (Ile47Thr and Val65Ala) . These mutations were absent in 144 normal ethnicity-matched controls, suggesting their pathogenicity in LHON within the context of the L3 haplogroup . This demonstrates how mutations may exert their effects through cumulative or haplogroup-specific mechanisms.

In comparative studies, researchers examine how MT-ND4L variants affect different species differently, potentially providing insights into species-specific adaptations or vulnerabilities. For instance, in Tetraodontiform fishes, studies have shown evidence of positive selection in various mitochondrial genes across different lineages, suggesting adaptive evolution . This comparative approach can help identify conserved functional domains versus regions under diverse selection pressures, informing our understanding of how mutations might contribute to disease or adaptive traits.

What methodological challenges exist in expressing and purifying functional recombinant MT-ND4L protein?

Expressing and purifying functional recombinant MT-ND4L presents several significant challenges due to its highly hydrophobic nature and the requirement for proper mitochondrial membrane integration. Key methodological challenges include:

• Expression system selection: The choice between prokaryotic and eukaryotic expression systems involves tradeoffs between yield, post-translational modifications, and proper folding. For Tetraodon nigroviridis MT-ND4L, researchers must empirically determine which system best preserves functional properties.

• Protein solubilization: As a highly hydrophobic membrane protein, MT-ND4L requires careful optimization of detergents or lipid mimetics to maintain structural integrity during purification.

• Codon optimization: Fish mitochondrial genes like those from Tetraodon nigroviridis often have codon usage different from standard expression hosts, necessitating codon optimization for efficient expression.

• Functional assessment: Validating that recombinant MT-ND4L maintains native enzymatic activity requires development of appropriate assays that can measure electron transport functionality in isolation or reconstituted systems.

• Complex assembly: MT-ND4L naturally functions as part of the large Complex I assembly, so researchers must decide whether to study the isolated subunit or attempt reconstitution with other complex components.

What insights can comparative analysis of MT-ND4L across fish species provide for understanding respiratory chain evolution?

Comparative analysis of MT-ND4L across fish species offers valuable insights into respiratory chain evolution, particularly in adaptation to different environmental conditions. Analysis of selection pressures on mitochondrial genes in Tetraodontiform fishes has revealed lineage-specific patterns of positive selection .

The table below shows the proportion of codons under significant positive selection in various genes across different fish lineages:

While this table doesn't specifically highlight MT-ND4L, it demonstrates the evolutionary patterns observed in related mitochondrial genes across Tetraodontiform lineages, which includes Tetraodon nigroviridis. These patterns suggest adaptation of the respiratory chain to different environmental pressures, diving depths, metabolic requirements, or other ecological factors across fish lineages.

What are the optimal protocols for expression and purification of functional recombinant Tetraodon nigroviridis MT-ND4L?

For optimal expression and purification of functional recombinant Tetraodon nigroviridis MT-ND4L, researchers should consider the following protocol framework:

• Gene synthesis and optimization:

  • Synthesize the MT-ND4L gene based on the Tetraodon nigroviridis mitochondrial genome sequence

  • Optimize codons for the chosen expression system

  • Add appropriate purification tags (His-tag or GST-tag) with protease cleavage sites

• Expression system selection:

  • For high yield: E. coli systems with specialized strains for membrane proteins (C41/C43)

  • For proper folding: Insect cell (Sf9, Sf21) or yeast (Pichia pastoris) systems that better handle membrane proteins

• Expression conditions:

  • Use lower temperatures (16-20°C) to slow folding and improve proper membrane insertion

  • Include membrane-mimetic compounds in growth media

  • Induce with lower concentrations of inducers for longer periods

• Membrane fraction isolation:

  • Gentle cell lysis using French press or sonication

  • Differential centrifugation to isolate membrane fractions

  • Careful solubilization using appropriate detergents (DDM, LMNG, or digitonin)

• Purification strategy:

  • Two-step purification using affinity chromatography followed by size exclusion

  • Maintain detergent above critical micelle concentration throughout purification

  • Consider lipid supplementation to maintain protein stability

• Functional validation:

  • NADH dehydrogenase activity assays

  • Reconstitution with other Complex I components

  • Electron microscopy to verify structural integrity

This methodological framework requires optimization for each specific research application, with particular attention to maintaining the native-like environment of this hydrophobic protein.

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

To effectively study interactions between recombinant Tetraodon nigroviridis MT-ND4L and other Complex I components, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Express tagged MT-ND4L in a heterologous system

  • Use antibodies against the tag to pull down MT-ND4L and associated proteins

  • Identify interaction partners using mass spectrometry

  • Verify specificity with appropriate controls including tag-only constructs

• Crosslinking coupled with mass spectrometry (XL-MS):

  • Use chemical crosslinkers with different spacer arms to capture transient or stable interactions

  • Digest crosslinked complexes and identify peptides by mass spectrometry

  • Map interaction interfaces at amino acid resolution

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize purified MT-ND4L on sensor chips or tips

  • Measure binding kinetics with purified partner proteins

  • Determine association/dissociation rates and binding affinities

• Reconstitution assays:

  • Systematically combine purified components to rebuild partial or complete Complex I

  • Measure activity as components are added to identify functional interactions

  • Use proteoliposomes to create more native-like membrane environments

• Cryo-electron microscopy:

  • Visualize assembled complexes containing MT-ND4L and binding partners

  • Generate 3D structural models of interaction interfaces

  • Compare structures with and without MT-ND4L to identify conformational changes

• Computational docking and molecular dynamics:

  • Generate structural models of MT-ND4L and interaction partners

  • Predict binding interfaces through docking simulations

  • Validate predictions through site-directed mutagenesis of key residues

This multi-method approach provides complementary data that together build a comprehensive understanding of MT-ND4L's role in Complex I assembly and function.

How should researchers interpret evolutionary selection patterns in MT-ND4L across different fish lineages?

When interpreting evolutionary selection patterns in MT-ND4L across fish lineages, researchers should consider multiple analytical frameworks:

• dN/dS ratio analysis:

  • Calculate the ratio of non-synonymous to synonymous substitutions (dN/dS)

  • dN/dS > 1 indicates positive selection; dN/dS < 1 indicates purifying selection

  • Site-specific models can identify specific amino acids under selection

  • Branch-site models can detect selection in specific lineages

• Structural mapping of selected sites:

  • Map selected residues onto structural models of MT-ND4L

  • Determine if selected sites cluster in functional domains, binding interfaces, or transmembrane regions

  • Evaluate potential functional impacts based on location

• Lineage-specific patterns:

  • Compare selection patterns between fish families with different ecological niches

  • Consider that different Tetraodontiform lineages (including Tetraodontidae, which contains Tetraodon nigroviridis) show different patterns of selection in mitochondrial genes, as seen in the data table from search result

  • Correlate selection patterns with environmental factors like temperature, pressure, or metabolic demands

• Functional validation:

  • Design experiments to test the functional consequences of selected residues

  • Use site-directed mutagenesis to introduce ancestral or derived states

  • Measure effects on enzyme activity, complex assembly, or proton pumping

• Haplogroup context:

  • Consider how mitochondrial background (haplogroup) affects the interpretation of selection

  • Draw parallels with human studies showing haplogroup effects on MT-ND4L mutations, as seen in the LHON study

• Convergent evolution:

  • Identify instances where similar selection patterns occur in unrelated lineages

  • Evaluate whether these represent adaptive responses to similar environmental challenges

This multifaceted interpretation approach provides a more complete picture of how MT-ND4L has evolved across fish lineages and helps distinguish between neutral variation, adaptive evolution, and functional constraints.

What analytical approaches are most effective for assessing the functional impact of MT-ND4L variants?

For assessing the functional impact of MT-ND4L variants, researchers should employ a comprehensive analytical strategy combining:

• Biochemical characterization:

  • Compare NADH dehydrogenase activity between wild-type and variant proteins

  • Measure electron transfer rates to ubiquinone

  • Assess complex assembly efficiency

  • Determine proton pumping capabilities

  • Quantify reactive oxygen species (ROS) production as a measure of electron leakage

• Structural analysis:

  • Use molecular dynamics simulations to predict structural perturbations

  • Apply circular dichroism (CD) to assess secondary structure changes

  • Employ hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • If possible, obtain cryo-EM structures of variant proteins within Complex I

• Cellular physiological assays:

  • Measure mitochondrial membrane potential in cells expressing variants

  • Assess cellular respiration using oxygen consumption rate (OCR) measurements

  • Evaluate ATP production capacity

  • Analyze mitochondrial morphology and network dynamics

  • Test cell viability under metabolic stress conditions

• In vivo models:

  • Generate transgenic models expressing Tetraodon nigroviridis MT-ND4L variants

  • Assess phenotypic outcomes including growth, development, and stress responses

  • Evaluate tissue-specific effects, particularly in high-energy demanding tissues

• Comparative evolutionary approach:

  • Draw parallels with known pathogenic variants in other species

  • Consider the disease-associated mutations found in human MT-ND4L, such as those linked to LHON with mutations 10609T>C and 10663T>C that lead to Ile47Thr and Val65Ala amino acid changes

  • Assess whether variants occur at conserved sites across species

• Integration with system-level data:

  • Combine functional assays with transcriptomic, proteomic, and metabolomic data

  • Develop network models of how MT-ND4L variants affect mitochondrial and cellular function

  • Quantify compensatory responses that may mask primary defects

This integrated analytical approach provides a comprehensive assessment of how MT-ND4L variants impact function at molecular, cellular, and organismal levels.

How does the structure and function of MT-ND4L in Tetraodon nigroviridis compare with other fish species and mammals?

The structure and function of MT-ND4L in Tetraodon nigroviridis compared to other species represents an important area for comparative research:

• Sequence conservation and divergence:

  • The human MT-ND4L gene produces a small 11 kDa protein composed of 98 amino acids

  • While specific differences in Tetraodon nigroviridis MT-ND4L are not detailed in the search results, comparative analysis typically reveals domains of high conservation (functional cores) versus regions with higher variability

  • Transmembrane domains tend to be more conserved than loop regions

  • Conservation analysis across diverse fish species and mammals can identify universally conserved residues likely essential for basic function

• Structural adaptations:

  • Tetraodon nigroviridis, as a pufferfish species, may exhibit adaptations related to its unique physiology

  • Adaptations might include changes in hydrophobicity patterns, charge distribution, or interaction surfaces

  • These adaptations could correlate with differences in mitochondrial membrane composition across species

• Complex I assembly:

  • The unusual gene overlap between MT-ND4L and MT-ND4 observed in humans may be conserved or modified in Tetraodon nigroviridis

  • Assembly mechanisms and interaction partners may differ between fish and mammalian systems

  • Species-specific chaperones or assembly factors might exist

• Functional differences:

  • Kinetic properties of Complex I containing Tetraodon nigroviridis MT-ND4L may be adapted to different temperature ranges

  • Efficiency of proton pumping and NADH oxidation might vary based on metabolic requirements

  • Susceptibility to inhibitors could differ between species

• Evolutionary rate:

  • Evidence from Tetraodontiform fishes shows variable selection pressure across mitochondrial genes in different lineages

  • This suggests lineage-specific adaptation of mitochondrial function, potentially including MT-ND4L

  • Understanding these differences can provide insights into environmental adaptations

This comparative approach helps identify both universal aspects of MT-ND4L function and species-specific adaptations, informing both basic research and potential biomedical applications.

What can mutational studies of recombinant MT-ND4L teach us about mitochondrial disease mechanisms?

Mutational studies of recombinant MT-ND4L provide critical insights into mitochondrial disease mechanisms:

• Structure-function relationships:

  • Systematic mutation of conserved residues can map functional domains

  • Studies of disease-associated mutations such as those found in LHON patients (10609T>C and 10663T>C resulting in Ile47Thr and Val65Ala) can reveal how specific amino acid changes disrupt function

  • The recombinant system allows direct comparison between wild-type and mutant proteins

• Pathogenic mechanisms:

  • Mutations may affect multiple aspects of MT-ND4L function:
    a. Complex I assembly efficiency
    b. Electron transfer capacity
    c. Proton pumping
    d. Reactive oxygen species production
    e. Protein stability and turnover

  • Biochemical characterization can determine which mechanisms predominate for specific mutations

• Threshold effects:

  • Mitochondrial diseases often exhibit threshold effects where a certain level of dysfunction must be reached before clinical manifestation

  • Mutational studies with recombinant proteins can establish dose-response relationships

  • Mixing different proportions of wild-type and mutant proteins can model heteroplasmy (mixed populations of normal and mutant mitochondrial DNA)

• Species-specific effects:

  • Comparing the same mutations in MT-ND4L from different species (human vs. Tetraodon nigroviridis) can reveal contextual factors

  • Some mutations may be pathogenic in one species but neutral in another due to different genetic backgrounds

  • The study of L3 haplogroup-specific effects in LHON demonstrates this principle in humans

• Therapeutic insights:

  • Understanding precisely how mutations disrupt function guides rational therapeutic development

  • Some defects might be amenable to small molecule intervention

  • Others might require gene therapy or protein replacement approaches

• Evolutionary medicine:

  • Recombinant systems allow testing of ancestral states and evolutionary intermediates

  • This approach can reveal why certain mutations are pathogenic and others are tolerated

  • It may explain why some regions of MT-ND4L are under strong purifying selection while others show evidence of positive selection in certain lineages

These mutational studies bridge the gap between clinical observations and molecular mechanisms, ultimately improving our understanding and management of mitochondrial diseases.

What emerging technologies will advance our understanding of MT-ND4L function and dysfunction?

Several emerging technologies promise to significantly advance our understanding of MT-ND4L:

• Cryo-electron microscopy advancements:

  • Improved resolution now allows visualization of individual side chains

  • Time-resolved cryo-EM can potentially capture different conformational states during catalysis

  • These advances will provide unprecedented insights into how MT-ND4L contributes to Complex I structure and function

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) applied to reconstituted systems

  • Optical tweezers to measure forces during conformational changes

  • These approaches can reveal dynamic aspects of MT-ND4L function not accessible to ensemble methods

• Mitochondrial genome editing:

  • CRISPR-based approaches adapted for mitochondrial DNA

  • Base editors and prime editors designed for mitochondrial targets

  • These tools will allow precise genetic manipulation of MT-ND4L in cellular and organismal contexts

• Artificial intelligence applications:

  • Improved protein structure prediction using AlphaFold-like approaches

  • Machine learning to identify patterns in functional data

  • Network analysis to understand system-level responses to MT-ND4L perturbations

• Advanced imaging technologies:

  • Super-resolution microscopy of tagged MT-ND4L in living cells

  • Correlative light and electron microscopy to link function to structure

  • These methods will bridge molecular and cellular levels of analysis

• In vitro mitochondrial systems:

  • Reconstituted minimal mitochondrial systems

  • Mitochondrial-on-a-chip technologies

  • These platforms will enable controlled studies of MT-ND4L in near-native environments

• Computational simulations:

  • Quantum mechanical/molecular mechanical (QM/MM) simulations of electron transfer

  • Coarse-grained approaches to model longer timescale processes

  • These computational approaches will provide insights into mechanisms difficult to access experimentally

These technologies, individually and in combination, will transform our understanding of this small but critical component of the mitochondrial respiratory chain.

How might comparative studies of MT-ND4L in Tetraodon nigroviridis inform therapeutic approaches for mitochondrial diseases?

Comparative studies of MT-ND4L in Tetraodon nigroviridis could inform therapeutic approaches for mitochondrial diseases in several innovative ways:

• Natural compensatory mechanisms:

  • Identifying how different species compensate for potentially harmful MT-ND4L variants

  • Understanding why mutations pathogenic in humans might be tolerated in fish

  • These insights could reveal natural protective mechanisms that could be therapeutically mimicked

• Small molecule discovery:

  • Screening for compounds that specifically interact with or stabilize fish MT-ND4L

  • Testing if these compounds can rescue human MT-ND4L variants

  • Comparative pharmacology between species can identify new therapeutic chemical spaces

• Protein engineering approaches:

  • Creating chimeric proteins incorporating resilient domains from fish MT-ND4L

  • Developing optimized MT-ND4L proteins with enhanced stability or catalytic properties

  • These engineered proteins could inform gene therapy approaches

• Environmental adaptations:

  • Understanding how Tetraodon nigroviridis MT-ND4L adapts to environmental stressors

  • Identifying if certain environmental conditions (temperature, oxygen levels) mitigate dysfunction

  • These insights could inform lifestyle or environmental interventions for patients

• Evolutionary medicine insights:

  • Reconstructing the evolutionary history of disease-associated residues

  • Understanding why certain variants became fixed in some lineages but cause disease in others

  • This evolutionary context helps distinguish truly dysfunctional variants from those that might be benign in certain contexts

• Bypass mechanisms:

  • Studying how different species cope with varying levels of Complex I activity

  • Identifying alternative electron transport pathways that could be therapeutically induced

  • These natural bypass strategies could inspire metabolic interventions

• Biomarkers and diagnostics:

  • Comparative studies may reveal conserved consequences of MT-ND4L dysfunction

  • These conserved features could serve as biomarkers for disease progression or therapeutic response

  • Improved diagnostics would enhance clinical trial design and patient stratification

The evolutionary distance between humans and Tetraodon nigroviridis provides a valuable comparative lens that can reveal both universal principles and creative solutions to mitochondrial dysfunction, potentially opening new therapeutic avenues.