Recombinant Ovis canadensis NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

How does the structure of MT-ND4L relate to its function in Complex I?

The structure of MT-ND4L is characterized by multiple transmembrane domains that anchor it within the inner mitochondrial membrane. As a core component of Complex I, MT-ND4L is positioned strategically to facilitate electron transfer from NADH to ubiquinone . Its membrane-embedded nature allows it to participate in proton translocation across the mitochondrial membrane, contributing to the establishment of the proton gradient necessary for ATP synthesis.

Recent structural analyses of similar proteins suggest that MT-ND4L interacts closely with other subunits of Complex I, including ND2, ND3, ND4, ND5, and ND6, with interaction scores approaching 0.999 according to STRING database analysis . These high-confidence protein-protein interactions indicate the essential role of MT-ND4L in maintaining the structural integrity and functional capacity of Complex I.

What are the optimal storage and handling conditions for recombinant MT-ND4L protein?

For optimal preservation of recombinant MT-ND4L protein activity:

• Store lyophilized protein at -20°C to -80°C for long-term storage .

• After reconstitution, prepare working aliquots supplemented with 5-50% glycerol (50% glycerol is recommended for optimal preservation) .

• Store working aliquots at 4°C for up to one week to maintain stability .

• Avoid repeated freeze-thaw cycles as this significantly decreases protein activity .

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

• For storage buffers, Tris/PBS-based buffers with 6% trehalose at pH 8.0 have shown optimal preservation of protein structure and function .

Following these guidelines will help ensure experimental reproducibility and maintain the structural integrity of the recombinant protein.

What experimental approaches are recommended for studying MT-ND4L interactions with other Complex I components?

Several methodological approaches are effective for studying MT-ND4L interactions:

• Immunoprecipitation coupled with mass spectrometry:

  • Use antibodies specific to MT-ND4L to pull down the protein complex

  • Identify interacting partners through mass spectrometric analysis

  • Verify interactions through reciprocal immunoprecipitation

• Proximity-dependent biotin labeling (BioID or APEX):

  • Generate fusion constructs of MT-ND4L with biotin ligase

  • Identify proteins in close proximity through streptavidin pulldown and mass spectrometry

  • This approach is particularly valuable for membrane proteins like MT-ND4L

• Two-site sandwich ELISA:

  • As described in the chicken MT-ND4L ELISA kit methodology, this approach can quantify MT-ND4L in biological samples

  • Pre-coat a microplate with an antibody specific for MT-ND4L

  • Add standards and samples to allow binding of MT-ND4L

  • Apply a biotin-conjugated antibody followed by streptavidin-HRP

  • Measure color development proportional to MT-ND4L concentration

• Blue native PAGE followed by immunoblotting:

  • This technique preserves protein complexes in their native state

  • Can reveal associations between MT-ND4L and other Complex I components

For all these methodologies, proper controls and validation experiments are essential for accurate interpretation of results.

How does Ovis canadensis MT-ND4L compare with MT-ND4L from other species, and what insights can be gained from these comparisons?

Comparative analysis of MT-ND4L across species reveals important evolutionary patterns:

When designing experiments using recombinant Ovis canadensis MT-ND4L, researchers should consider these species-specific variations, especially when extrapolating findings to other organisms.

What role does MT-ND4L play in high-altitude adaptation in species like Tibetan yaks and cattle?

MT-ND4L has been implicated in high-altitude adaptation through genetic diversity studies. Research on Tibetan yaks, Tibetan cattle, and Holstein-Friesian cattle has revealed that:

These findings highlight MT-ND4L as a potential genetic marker for high-altitude adaptation and suggest that further research into the functional consequences of these genetic variations could provide insights into mitochondrial adaptations to hypoxic conditions .

What is the evidence linking MT-ND4L mutations to Leber hereditary optic neuropathy (LHON)?

A specific mutation in MT-ND4L has been identified in several families with Leber hereditary optic neuropathy (LHON). This mutation is characterized as:

• Molecular designation: T10663C or Val65Ala

• Consequence: Substitution of valine with alanine at position 65 in the protein sequence

• Clinical significance: Associated with the characteristic vision loss of LHON

• Disruption of electron transfer efficiency within Complex I

• Increased production of reactive oxygen species

• Compromised ATP production in retinal ganglion cells

• Altered interactions with other Complex I subunits, particularly ND3, ND4, and ND6

Research approaches to better understand this relationship include:

• Generation of cellular models expressing the Val65Ala mutation

• Assessment of Complex I activity in patient-derived samples

• Evaluation of mitochondrial membrane potential in affected tissues

• Measurement of ROS production and oxidative stress markers

How is MT-ND4L involved in mitochondrial dysfunction related to atherosclerosis and metabolic disorders?

Research has demonstrated connections between mitochondrial DNA damage, particularly affecting genes like MT-ND4L, and the development of atherosclerosis and metabolic disorders:

• Mitochondrial DNA is particularly vulnerable to damage due to:

  • Lack of protective histones

  • Proximity to the inner mitochondrial membrane

  • Exposure to reactive oxygen species generated during oxidative phosphorylation

• Damage to mitochondrial genes encoding Complex I subunits (including MT-ND4L) has several consequences:

  • Disruption of electron transport chain function

  • Increased ROS production creating a damaging feedback loop

  • Compromised ATP production

  • Activation of pro-inflammatory signaling pathways

• In atherosclerosis models, mitochondrial dysfunction involving Complex I has been linked to:

  • Accelerated plaque formation

  • Vascular smooth muscle cell dysfunction

  • Impaired macrophage function

  • Development of metabolic syndrome features including glucose intolerance

• Experimental approaches to study this connection include:

  • Assessment of mitochondrial deletion frequency

  • Measurement of Complex I activity in affected tissues

  • Evaluation of oxidative phosphorylation efficiency

  • Analysis of ROS production and oxidative damage markers

The relationship between MT-ND4L function, mitochondrial damage, and cardiovascular disease represents an important area for further investigation, with potential implications for therapeutic interventions targeting mitochondrial function.

What are the methodological considerations for investigating MT-ND4L mutations and their effects on Complex I assembly and function?

Advanced investigation of MT-ND4L mutations requires sophisticated methodological approaches:

• CRISPR-Cas9 Mitochondrial Gene Editing:

  • Recently developed techniques allow precise editing of mitochondrial genes

  • Can create isogenic cell lines differing only in MT-ND4L sequence

  • Enables direct assessment of mutation effects on Complex I function

• Blue Native PAGE for Complex I Assembly Analysis:

  • Preserves native protein complexes during electrophoresis

  • Can detect assembly intermediates and subcomplexes

  • Combined with Western blotting using subunit-specific antibodies

  • Quantitative assessment of fully assembled Complex I versus subcomplexes

• High-Resolution Respirometry:

  • Measures oxygen consumption in intact cells or isolated mitochondria

  • Can assess specific Complex I-dependent respiration using substrate combinations

  • Evaluates coupling efficiency through respiratory control ratios

  • Detects subtle functional defects not apparent with other techniques

• Cryo-EM Structural Analysis:

  • Provides atomic-level resolution of Complex I structure

  • Can visualize structural perturbations caused by MT-ND4L mutations

  • Enables mapping of mutation effects on subunit interactions

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Detects conformational changes in protein structure

  • Can identify regions of altered dynamics in mutant proteins

  • Provides insights into structural consequences of mutations

When investigating MT-ND4L mutations, it is critical to consider the appropriate cellular model, as nuclear genetic background can significantly influence the phenotypic expression of mitochondrial mutations through retrograde signaling pathways.

How can systems biology approaches be applied to understand the broader impact of MT-ND4L variations on cellular metabolism?

Systems biology offers powerful frameworks for understanding MT-ND4L's role in broader metabolic networks:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Map changes in metabolic pathways affected by MT-ND4L variations

  • Identify compensatory mechanisms and adaptive responses

  • Example approach: After introducing MT-ND4L mutations, perform RNA-seq, proteomic analysis, and untargeted metabolomics to create an integrated model of cellular response

• Flux Balance Analysis (FBA):

  • Mathematical modeling of metabolic networks

  • Predicts metabolic flux distributions under different conditions

  • Can incorporate MT-ND4L-mediated effects on respiratory chain function

  • Enables in silico prediction of metabolic consequences

• 13C Metabolic Flux Analysis:

  • Uses isotope-labeled substrates to trace metabolic pathways

  • Quantifies changes in TCA cycle flux, gluconeogenesis, and other pathways

  • Can directly measure consequences of Complex I dysfunction on central carbon metabolism

  • Particularly valuable for understanding compensatory metabolic rewiring

• In silico Modeling of Electron Transport Chain:

  • Computational models of electron flow through Complex I

  • Predicts functional consequences of specific amino acid substitutions

  • Can be validated experimentally through site-directed mutagenesis

• Network Analysis of Protein-Protein Interactions:

  • Based on STRING database interaction scores (which show very high confidence scores >0.99 for interactions between MT-ND4L and other Complex I subunits)

  • Identifies altered interaction networks resulting from MT-ND4L variants

  • Can predict secondary effects on mitochondrial function

These systems approaches are particularly valuable for understanding how relatively subtle changes in MT-ND4L structure or function can propagate through cellular metabolic networks to influence phenotypes ranging from high-altitude adaptation to pathological states.

What are the recommended analytical approaches for quantifying MT-ND4L protein and assessing its incorporation into Complex I?

Several analytical techniques are suitable for quantitative assessment of MT-ND4L:

• Quantitative Mass Spectrometry:

  • Selected Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM)

  • Targeted detection of specific MT-ND4L peptides

  • Can use isotope-labeled standards for absolute quantification

  • Enables simultaneous measurement of multiple Complex I subunits

• Sandwich ELISA:

  • As detailed in the chicken MT-ND4L ELISA kit methodology

  • Antibody-based capture and detection system

  • Sensitive quantification in complex biological samples

  • Standard curves typically range from picogram to nanogram levels

• Immunoblotting with Fluorescent Secondary Antibodies:

  • Enables linear quantification over a broader range than traditional chemiluminescence

  • Can simultaneously detect multiple Complex I subunits with appropriate antibodies

  • Allows normalization to total protein content

• Complex I In-Gel Activity Assays:

  • Following blue native PAGE separation

  • Uses NADH as substrate and nitrotetrazolium blue as electron acceptor

  • Produces purple precipitate at the site of Complex I

  • Can be quantified by densitometry

• Spectrophotometric Complex I Activity Assays:

  • Measures NADH oxidation rate at 340 nm

  • Can assess rotenone-sensitive activity (specific to Complex I)

  • Often normalized to citrate synthase activity as a mitochondrial content marker

  • Can detect functional impairments resulting from altered MT-ND4L incorporation

When selecting an analytical approach, researchers should consider the specific research question, sample type, required sensitivity, and available instrumentation.

How can researchers effectively analyze the impact of MT-ND4L variations on mitochondrial function and bioenergetics?

Comprehensive analysis of MT-ND4L's impact on mitochondrial function requires multiple complementary approaches:

• Oxygen Consumption Measurements:

  • High-resolution respirometry (e.g., Oroboros Oxygraph)

  • Seahorse XF analyzer for cellular oxygen consumption rate (OCR)

  • Assessment of specific respiratory states using substrate-inhibitor combinations

  • Calculation of respiratory control ratios and P/O ratios

• Membrane Potential Analysis:

  • Potentiometric dyes (TMRM, JC-1) for qualitative and semi-quantitative assessment

  • Safranin O for quantitative measurements in isolated mitochondria

  • Flow cytometry or confocal microscopy for single-cell analyses

  • Critical for detecting subtle defects in proton pumping capacity

• Superoxide and ROS Measurements:

  • MitoSOX for mitochondrial superoxide detection

  • DCF-DA for general cellular ROS

  • Amplex Red for hydrogen peroxide quantification

  • Essential for assessing oxidative stress resulting from Complex I dysfunction

• ATP Production Assays:

  • Luciferase-based assays for total cellular ATP

  • Measurements of ATP synthesis rates in isolated mitochondria

  • ATP/ADP ratio determination with specialized sensors

  • Directly addresses functional outcome of MT-ND4L variations

• Mitochondrial DNA Damage Assessment:

  • PCR-based methods for detecting mtDNA deletions (particularly the "common deletion")

  • Quantification of mtDNA copy number relative to nuclear DNA

  • Assessment of specific damage to mtDNA encoding Complex I subunits

• Metabolomic Profiling:

  • Targeted analysis of TCA cycle intermediates

  • Measurements of lactate/pyruvate ratios

  • Assessment of β-hydroxybutyrate levels

  • Identifies metabolic adaptations to Complex I dysfunction

When integrating these approaches, researchers can develop a comprehensive understanding of how variations in MT-ND4L structure or abundance impact the broader landscape of mitochondrial function and cellular bioenergetics.

What are the emerging areas of research involving MT-ND4L that hold promise for understanding mitochondrial diseases and adaptations?

Several cutting-edge research directions involving MT-ND4L show significant promise:

• Single-Cell Analysis of MT-ND4L Expression and Function:

  • Assessing cell-to-cell variability in MT-ND4L abundance

  • Correlating MT-ND4L levels with mitochondrial function at single-cell resolution

  • Understanding the heteroplasmy threshold effect in mitochondrial diseases

• Development of MT-ND4L-Targeted Therapeutics:

  • Peptide-based approaches to stabilize Complex I assembly

  • Small molecules that compensate for MT-ND4L mutations

  • Gene therapy approaches for LHON and other associated conditions

• MT-ND4L in Cellular Adaptation to Environmental Stressors:

  • Building on findings from high-altitude adaptation studies

  • Investigating MT-ND4L's role in hypoxic preconditioning

  • Exploring thermal and metabolic stress responses

• Interaction Between MT-ND4L Variants and Aging:

  • Role in accumulation of mtDNA damage over lifespan

  • Contribution to age-related mitochondrial dysfunction

  • Potential therapeutic target for age-related conditions

• Tissue-Specific Effects of MT-ND4L Variations:

  • Understanding why certain tissues (e.g., retinal ganglion cells in LHON) are particularly vulnerable

  • Mapping tissue-specific expression patterns and protein interactions

  • Developing tissue-targeted therapeutic approaches

These emerging areas represent promising directions for researchers seeking to advance understanding of MT-ND4L's role in health, disease, and evolutionary adaptation.

How might advanced computational and structural biology approaches enhance our understanding of MT-ND4L function in Complex I?

Advanced computational approaches offer powerful tools for MT-ND4L research:

• Molecular Dynamics Simulations:

  • Atomistic modeling of MT-ND4L within the Complex I structure

  • Prediction of conformational changes during catalytic cycle

  • Assessment of how mutations alter protein dynamics and interactions

  • Simulation of proton translocation mechanisms

• Machine Learning for Variant Effect Prediction:

  • Development of algorithms to predict functional consequences of MT-ND4L variants

  • Integration of structural, evolutionary, and functional data

  • Potential to identify previously unrecognized pathogenic variants

• Quantum Mechanical/Molecular Mechanical (QM/MM) Simulations:

  • High-precision modeling of electron transfer reactions

  • Understanding how MT-ND4L variations affect electron tunneling probabilities

  • Insights into fundamental bioenergetic mechanisms

• Cryo-EM Structural Analysis of Complex I States:

  • Capturing different conformational states during catalytic cycle

  • Visualizing how MT-ND4L participates in proton pumping

  • Structural basis for disease-associated mutations

• Integrative Structural Biology:

  • Combining data from multiple experimental approaches (crosslinking mass spectrometry, HDX-MS, FRET)

  • Creating comprehensive models of MT-ND4L interactions and dynamics

  • Predicting effects of mutations on Complex I assembly and function

By leveraging these advanced computational approaches, researchers can develop deeper insights into the structural basis of MT-ND4L function and dysfunction, potentially enabling rational design of therapeutic interventions for associated disorders.