Recombinant Lagorchestes hirsutus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is Lagorchestes hirsutus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)?

Lagorchestes hirsutus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L) is a mitochondrial protein that functions as part of Complex I in the respiratory chain. This protein is encoded by the MT-ND4L gene found in the mitochondrial genome of Lagorchestes hirsutus (Rufous hare-wallaby or Western hare-wallaby). It is a small membrane protein consisting of 98 amino acids and is officially classified with the enzyme commission number EC 1.6.5.3, also known as NADH dehydrogenase subunit 4L . The protein plays a crucial role in the electron transport process during oxidative phosphorylation, specifically in the transfer of electrons from NADH to ubiquinone, which is an essential step in cellular energy production.

What is the normal function of the MT-ND4L gene and protein?

The MT-ND4L gene provides instructions for making the NADH dehydrogenase 4L protein, which is an integral component of Complex I in the mitochondrial respiratory chain. This complex is embedded in the inner mitochondrial membrane and participates in oxidative phosphorylation, the process by which cells convert energy from food into ATP. Specifically, Complex I is responsible for the first step in electron transport, transferring electrons from NADH to ubiquinone . This electron transfer helps establish an electrochemical gradient across the inner mitochondrial membrane, creating an unequal electrical charge that provides the energy necessary for ATP production . The MT-ND4L protein contributes to the structural integrity and functional efficiency of Complex I, supporting this critical energy-generating process in cells.

How should recombinant MT-ND4L be stored and handled for optimal stability?

For optimal stability of recombinant Lagorchestes hirsutus MT-ND4L, researchers should follow specific storage and handling protocols. The protein should be stored in a Tris-based buffer containing 50% glycerol, which has been optimized specifically for this protein . Short-term storage should be at -20°C, while extended storage requires -20°C or preferably -80°C to maintain protein integrity. Working aliquots can be maintained at 4°C for up to one week to minimize freeze-thaw cycles . It is crucial to avoid repeated freezing and thawing, as this can lead to protein denaturation and loss of activity. When handling the protein, researchers should consider creating small working aliquots to minimize exposure to degradative conditions. Additionally, all buffers used should be free of contaminants that could affect protein stability or downstream applications, and appropriate protease inhibitors may be added if required for specific experimental setups.

What experimental approaches are suitable for studying MT-ND4L function in mitochondrial research?

Several experimental approaches are valuable for investigating MT-ND4L function:

• Complex I Activity Assays: Researchers can measure NADH:ubiquinone oxidoreductase activity using spectrophotometric methods to assess the functional impact of recombinant MT-ND4L in reconstituted systems or the effects of mutations.

• Genetic Manipulation Techniques: The MitoKO DdCBE (DddA-derived cytosine base editor) system has been specifically designed to introduce premature stop codons in mitochondrial genes, including MT-ND4L. For MT-ND4L, researchers have successfully changed a coding sequence for Val90 and Gln91 (GTC CAA) into Val and STOP (GTT TAA) . This approach enables specific knockout of MT-ND4L for functional studies.

• Structural Analysis: Using the AlphaFold-predicted structure (AF-Q1MWF2-F1) with its high confidence metrics (pLDDT score of 89.9), researchers can investigate structure-function relationships . This can be complemented with experimental techniques like cryo-EM for validation.

• AI-Driven Conformational Studies: Advanced AI algorithms can predict alternative functional states of MT-ND4L, including large-scale conformational changes. Molecular simulations with AI-enhanced sampling and trajectory clustering allow exploration of the protein's conformational space, identifying representative structures that capture its dynamic behavior .

• Binding Pocket Analysis: AI-based pocket prediction can discover orthosteric, allosteric, hidden, and cryptic binding pockets on MT-ND4L's surface, integrating literature search data with structure-aware ensemble-based detection algorithms .

What techniques can be used to study MT-ND4L protein interactions?

To study MT-ND4L protein interactions, researchers can employ several complementary techniques:

Table 1: Techniques for Studying MT-ND4L Protein Interactions

The AI-driven conformational ensemble generation approach described in the research allows for a more comprehensive understanding of MT-ND4L's dynamic behavior, providing insights into potential interaction sites that might not be apparent from static structures . This information can guide the design of more targeted experimental approaches to validate specific protein-protein interactions within the Complex I assembly.

How do mutations in MT-ND4L affect mitochondrial function and disease pathogenesis?

Mutations in the MT-ND4L gene can significantly impact mitochondrial function and contribute to disease pathogenesis. One well-documented mutation is T10663C (Val65Ala), which has been identified in several families with Leber hereditary optic neuropathy (LHON) . This mutation changes a single amino acid in the protein, replacing valine with alanine at position 65. The exact mechanism by which this mutation leads to the characteristic vision loss in LHON remains under investigation.

From a functional perspective, mutations in MT-ND4L can disrupt Complex I assembly or function in several ways:

• Reduced Complex I Activity: Analysis of knockouts has shown substantially reduced levels of Complex I in Nd4l (along with Nd5 and Nd6) knockouts . This reduction in Complex I activity can impair electron transport and decrease ATP production.

• Increased Reactive Oxygen Species (ROS): Dysfunction in Complex I due to MT-ND4L mutations may lead to electron leakage and increased production of ROS, causing oxidative stress and cellular damage.

• Altered Membrane Potential: Disruptions in the electron transport process can affect the establishment of the electrochemical gradient across the inner mitochondrial membrane, reducing the energy available for ATP synthesis.

• Tissue-Specific Effects: The impact of MT-ND4L mutations may vary across different tissues, explaining why conditions like LHON primarily affect specific cell types such as retinal ganglion cells despite the mutation being present in all cells containing mitochondria.

Understanding these mechanisms requires sophisticated research approaches, including the generation of disease models using techniques such as the MitoKO DdCBE system described in the literature , which allows for precise genetic manipulation of MT-ND4L to study the functional consequences of specific mutations.

How can gene editing techniques be applied to study MT-ND4L function?

Advanced gene editing techniques offer powerful tools for studying MT-ND4L function. The MitoKO DdCBE (DddA-derived cytosine base editor) system represents a significant breakthrough in this field, allowing researchers to introduce precise mutations in mitochondrial DNA, which was previously challenging due to the inaccessibility of mitochondrial genomes to CRISPR-Cas systems.

For MT-ND4L specifically, researchers have designed constructs that can change the coding sequence:

• Premature Stop Codon Introduction: While most mitochondrial genes can be targeted by changing Trp codons (TGA) into stop codons (TAA) by deaminating the C on the non-coding strand, MT-ND4L required a different approach. Researchers successfully changed a coding sequence for Val90 and Gln91 (GTC CAA) into Val and STOP (GTT TAA) .

• TALE-Based Targeting: The system employs TALEs (Transcription Activator-Like Effectors) that bind specifically to either the light (L) or heavy (H) strands of mitochondrial DNA. For optimal editing efficiency, researchers tested different combinations of the 1333 DddAtox split orientation .

• Transfection and Analysis Protocol: After transfecting these constructs into cells (e.g., NIH/3T3 mouse cells), researchers used FACS at 24 hours post-transfection to enrich the population of cells expressing the designated MitoKO DdCBEs .

This approach allows for:

• Creation of MT-ND4L knockout models to study loss-of-function effects

• Introduction of specific disease-associated mutations to create disease models

• Investigation of structure-function relationships by targeted modification of specific domains

• Study of MT-ND4L's contribution to Complex I assembly and function

By comparing the phenotypes of wild-type and edited cells/organisms, researchers can gain valuable insights into the role of MT-ND4L in mitochondrial function and disease pathogenesis.

What are the challenges in expressing and purifying functional recombinant MT-ND4L?

Expressing and purifying functional recombinant MT-ND4L presents several significant challenges due to its nature as a small, hydrophobic mitochondrial membrane protein. Researchers face the following obstacles:

Table 2: Challenges in Recombinant MT-ND4L Expression and Purification

The commercially available recombinant protein is supplied in a specialized storage buffer (Tris-based buffer with 50% glycerol) that has been optimized specifically for this protein , highlighting the importance of buffer composition for maintaining stability. Additionally, the tag type used for purification "will be determined during production process" , suggesting that different tagging strategies may need to be evaluated to optimize expression and purification for each batch.

Advanced structural prediction methods, such as those employed in AlphaFold to generate the AF-Q1MWF2-F1 model , can help guide expression and purification strategies by providing insights into the protein's structural features and potential stabilizing interactions.

How are mutations in MT-ND4L linked to Leber hereditary optic neuropathy (LHON)?

Leber hereditary optic neuropathy (LHON) is a mitochondrial genetic disorder characterized by the degeneration of retinal ganglion cells and their axons, leading to acute or subacute vision loss. A specific mutation in the MT-ND4L gene has been identified in several families with LHON, providing evidence for its role in disease pathogenesis.

The mutation in question is T10663C, which results in the amino acid substitution Val65Ala (valine to alanine at position 65) in the NADH dehydrogenase 4L protein . This single amino acid change occurs within a critical region of the protein that may affect its function or interaction with other subunits of Complex I.

The pathophysiological mechanism linking this mutation to LHON remains incompletely understood, but several hypotheses have been proposed:

• Impaired Complex I Function: The mutation may compromise the electron transfer efficiency of Complex I, reducing ATP production in affected cells.

• Increased Oxidative Stress: Dysfunction in Complex I can lead to increased production of reactive oxygen species (ROS), causing oxidative damage particularly in the highly metabolically active retinal ganglion cells.

• Tissue-Specific Energy Deficiency: The mutation may create a subtle energy deficiency that is particularly detrimental to retinal ganglion cells due to their high energy demands and unique structural features (unmyelinated portion of axons).

• Apoptotic Signaling: Disrupted mitochondrial function may trigger apoptotic pathways in retinal ganglion cells, leading to their degeneration.

Research tools such as the MitoKO system could be valuable for creating cellular models with this specific mutation to further investigate the molecular mechanisms. Additionally, AI-driven analysis as described in some research approaches could help predict the structural and functional impacts of this mutation on protein dynamics and interactions within Complex I.

What experimental models are suitable for studying MT-ND4L-related diseases?

Several experimental models can be employed to study MT-ND4L-related diseases, each with specific advantages for investigating different aspects of pathophysiology:

Table 3: Experimental Models for MT-ND4L-Related Disease Research

The MitoKO system described in the literature represents a significant advancement in creating precise mitochondrial gene knockouts or mutations . For MT-ND4L specifically, researchers have successfully designed constructs that can change the coding sequence for Val90 and Gln91 (GTC CAA) into Val and STOP (GTT TAA), enabling the creation of knockout models. This approach allows for detailed investigation of how MT-ND4L dysfunction contributes to disease pathogenesis.

Additionally, the AI-based characterization approaches mentioned in some research can provide valuable insights into how specific mutations might affect protein structure and function . By integrating these computational predictions with experimental validation in the models described above, researchers can develop a more comprehensive understanding of the molecular mechanisms underlying MT-ND4L-related diseases.

What controls should be included in MT-ND4L functional studies?

When conducting functional studies of MT-ND4L, incorporating appropriate controls is essential for ensuring experimental validity and accurate interpretation of results. The following controls should be considered:

Positive Controls:

• Wild-type MT-ND4L: Include unmodified MT-ND4L to establish baseline functional parameters.

• Known Functional Variants: Include MT-ND4L variants with well-characterized functional properties to validate assay sensitivity.

• Complex I Activity Standards: Use purified Complex I or submitochondrial particles with established activity levels as reference standards for functional assays.

Negative Controls:

• Empty Vector Controls: For expression studies, include cells transfected with empty vectors to account for transfection effects.

• Inactive Mutants: Include MT-ND4L variants with mutations known to abolish function, such as the STOP codon introduction described in the MitoKO system .

• Complex I Inhibitors: Use specific inhibitors (e.g., rotenone) as chemical negative controls in functional assays.

Technical Controls:

• Loading Controls: For western blots or other protein analysis methods, include mitochondrial or cellular housekeeping proteins.

• Mitochondrial Mass Markers: Include markers that control for differences in mitochondrial content between samples.

• Buffer-Only Controls: Include buffer-only samples to account for background signals in activity assays.

Specificity Controls:

• Other Complex I Subunits: Include studies of other Complex I subunits to distinguish MT-ND4L-specific effects from general Complex I dysfunction.

• Rescue Experiments: Attempt to rescue phenotypes by reintroducing wild-type MT-ND4L in knockout or mutant models.

• Off-Target Effect Controls: For gene editing approaches like the MitoKO system , include controls to assess potential off-target effects.

How can researchers quantify MT-ND4L expression levels in experimental systems?

Quantifying MT-ND4L expression levels presents unique challenges due to its small size, hydrophobic nature, and mitochondrial localization. Researchers can employ several complementary approaches:

• qRT-PCR for mRNA Quantification:

  • Design primers specific to MT-ND4L transcript

  • Normalize to mitochondrial housekeeping genes

  • Account for mitochondrial copy number variation

• Western Blot Analysis:

  • Use specialized protocols for small hydrophobic proteins

  • Select appropriate sample preparation methods to prevent aggregation

  • Choose loading controls specific to mitochondrial proteins

• Mass Spectrometry-Based Quantification:

  • Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM)

  • Use stable isotope-labeled peptide standards

  • Optimize sample preparation for membrane proteins

• Immunocytochemistry/Immunohistochemistry:

  • Assess relative expression levels in different cell types or tissues

  • Co-stain with mitochondrial markers

  • Use confocal microscopy for co-localization studies

• Blue Native PAGE:

  • Evaluate MT-ND4L incorporation into assembled Complex I

  • Assess relative abundance compared to other Complex I subunits

  • Combine with western blotting for specific detection

• Next-Generation Sequencing Approaches:

  • RNA-Seq with mitochondrial transcript enrichment

  • Analyze MT-ND4L transcript levels relative to other mitochondrial genes

  • Assess heteroplasmy levels in mutant models

When working with recombinant MT-ND4L protein, researchers should consider developing standard curves using the purified protein (such as the commercially available 50 μg preparation ) for accurate quantification. Additionally, the sophisticated AI-driven analysis methods described in some research approaches might be adapted to develop more sensitive detection methods for this challenging protein.

What are the considerations for designing MT-ND4L knockdown/knockout experiments?

Designing effective MT-ND4L knockdown/knockout experiments requires careful consideration of several factors due to the unique challenges associated with manipulating mitochondrial genes:

• Selection of Gene Editing Technology:

  • The MitoKO DdCBE system has been specifically designed for mitochondrial gene editing and has been successfully applied to MT-ND4L

  • For MT-ND4L, researchers changed a coding sequence for Val90 and Gln91 (GTC CAA) into Val and STOP (GTT TAA)

  • Consider the orientation of DddAtox splits and TALE binding domains for optimal editing efficiency

• Validation of Knockdown/Knockout Efficiency:

  • Assess MT-ND4L protein levels using western blot or mass spectrometry

  • Measure MT-ND4L transcript levels using qRT-PCR

  • Evaluate Complex I assembly using blue native PAGE

  • Confirm functional consequences through Complex I activity assays

• Control for Off-Target Effects:

  • Perform whole mitochondrial genome sequencing to identify potential off-target mutations

  • Include mock-transfected controls and non-targeting controls

  • Design rescue experiments with wild-type MT-ND4L expression

• Phenotypic Analysis:

  • Measure mitochondrial membrane potential

  • Assess oxygen consumption rates

  • Quantify ATP production

  • Evaluate ROS production

  • Examine cell viability and growth rates

• Experimental Timeline Considerations:

  • Allow sufficient time for turnover of pre-existing MT-ND4L protein

  • Consider the stability of assembled Complex I after MT-ND4L depletion

  • Plan for potential compensatory mechanisms that may develop over time

• Cell Type Selection:

  • Choose cell types with high mitochondrial content for more pronounced phenotypes

  • Consider tissue-specific effects relevant to diseases like LHON

  • Compare results across multiple cell types to assess generalizability

By carefully addressing these considerations, researchers can design robust experiments to investigate the functional consequences of MT-ND4L depletion and gain insights into its role in normal physiology and disease pathogenesis.