Recombinant Metachirus nudicaudatus 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 gene encoded by the mitochondrial genome that produces a critical subunit of NADH dehydrogenase (Complex I) of the electron transport chain. This protein functions as part of the largest of the five respiratory complexes and is highly hydrophobic in nature. The MT-ND4L protein specifically contributes to the core of the transmembrane region of Complex I, which is essential for proton pumping across the inner mitochondrial membrane during oxidative phosphorylation. In Metachirus nudicaudatus, as in other mammals, this protein plays a fundamental role in energy production through the electron transport chain. The protein consists of 98 amino acids and weighs approximately 11 kDa, maintaining a similar structure to MT-ND4L proteins found in other mammalian species .

How does MT-ND4L compare between Metachirus nudicaudatus and other species?

Comparative analysis of MT-ND4L sequences reveals evolutionary conservation across mammalian species while highlighting specific adaptations. Below is a comparison between Metachirus nudicaudatus (Brown four-eyed opossum) and Bos mutus grunniens (Wild yak) MT-ND4L sequences:

Both proteins maintain the same length (98 amino acids) and share similar hydrophobic properties essential for their transmembrane localization. The conservation of length across species from marsupials to placental mammals suggests strict evolutionary constraints on MT-ND4L structure due to its critical role in mitochondrial function .

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

For optimal stability and activity of recombinant Metachirus nudicaudatus MT-ND4L, researchers should store the protein at -20°C in a Tris-based buffer containing 50% glycerol. For extended storage periods, maintaining the protein at -80°C is recommended. To minimize protein degradation, avoid repeated freeze-thaw cycles which can compromise structural integrity and biological activity. Instead, prepare small working aliquots that can be stored at 4°C for up to one week. The storage buffer is specifically optimized for this hydrophobic membrane protein to prevent aggregation and maintain proper folding. Researchers should validate protein stability after storage using activity assays or structural analysis methods appropriate for membrane proteins .

How can researchers effectively study MT-ND4L interactions with other Complex I subunits?

Studying MT-ND4L interactions with other Complex I subunits requires specialized approaches due to its hydrophobic nature and transmembrane localization. Researchers should consider the following methodological strategy:

• Crosslinking studies: Use membrane-permeable crosslinkers followed by mass spectrometry to identify interaction partners within the complex.

• Blue native PAGE: Employ this technique to isolate intact Complex I while preserving native interactions between subunits.

• Co-immunoprecipitation with specialized detergents: Use mild detergents like digitonin or DDM (n-dodecyl β-D-maltoside) that preserve membrane protein interactions.

• Proximity labeling approaches: Methods such as BioID or APEX2 can identify proteins in close proximity to MT-ND4L within the mitochondrial membrane.

• Computational modeling: Leverage existing structural data from related species to predict interaction interfaces specific to Metachirus nudicaudatus MT-ND4L.

It's important to note that MT-ND4L forms the core of the transmembrane region of Complex I, making it particularly challenging to study in isolation while maintaining its native conformation and interaction capacity .

What gene editing approaches can be used to study MT-ND4L function?

Recent advances in mitochondrial gene editing provide powerful tools for functional studies of MT-ND4L. A notable approach involves DddA-derived cytosine base editors (DdCBEs) that can introduce specific mutations in mitochondrial DNA:

• Targeted STOP codon introduction: For most mitochondrial ORFs, researchers have successfully used base editing to convert Trp codons (TGA) into stop codons (TAA) by deaminating specific cytosines on the non-coding strand.

• MT-ND4L-specific approach: Due to its sequence characteristics, a different strategy is employed for MT-ND4L. Researchers have converted a sequence coding for Val90 and Gln91 (GTC CAA) into Val and STOP (GTT TAA) through targeted cytosine deamination.

• Sequential transfection protocol: To achieve high levels of mtDNA editing, researchers can implement multiple rounds of transfection and recovery. This involves:

  • Delivery of MitoKO constructs

  • Selection of transfectants by FACS at 24 hours post-transfection

  • A 14-day recovery period

  • Heteroplasmy measurement

  • Repeated transfection

This iterative approach has successfully generated effectively homoplasmic cells harboring premature stop codons in all mouse mtDNA-encoded protein-coding genes, including MT-ND4L .

What is the significance of the MT-ND4L variant rs28709356 in Alzheimer's disease research?

The MT-ND4L variant rs28709356 (C>T) has emerged as a significant genetic factor in Alzheimer's disease (AD) research based on whole exome sequencing data analysis. A comprehensive study involving 10,831 participants from the Alzheimer's Disease Sequencing Project revealed several important findings:

• Statistical significance: Analysis of 4,220 mtDNA variants showed a study-wide significant association between AD and the rare MT-ND4L variant rs28709356 (p = 7.3 × 10^-5).

• Rarity of the variant: The minor allele frequency was determined to be 0.002, indicating its rare occurrence in the population.

• Gene-based testing: Beyond the single variant association, MT-ND4L as a whole showed significant association with AD in gene-based tests (p = 6.71 × 10^-5).

• Mitochondrial dysfunction hypothesis: These findings provide compelling evidence supporting the role of mitochondrial dysfunction in AD pathogenesis, specifically implicating Complex I components.

Researchers studying MT-ND4L should consider screening for this variant when investigating potential links between mitochondrial function and neurodegenerative disorders. The data suggests that even rare variants in MT-ND4L can have significant implications for disease susceptibility, highlighting the importance of comprehensive genetic analysis in mitochondrial research .

How does the unusual gene overlap between MT-ND4L and MT-ND4 impact experimental design?

The unusual 7-nucleotide gene overlap between MT-ND4L and MT-ND4 presents distinct challenges for experimental design that researchers must carefully address:

• Primer design considerations: When designing primers for PCR amplification or sequencing of either gene, researchers must consider the overlapping region to avoid unintended amplification or interference.

• Mutagenesis strategies: Any targeted mutagenesis of the 3' end of MT-ND4L must account for potential effects on MT-ND4 expression, as modifications could affect both genes simultaneously.

• Transcript analysis complexity: RNA-based studies require careful design to distinguish between MT-ND4L and MT-ND4 transcripts, particularly when analyzing processing and maturation of mitochondrial RNAs.

• Reading frame awareness: The MT-ND4 gene starts in the +3 reading frame with respect to the MT-ND4L reading frame, creating a complex genetic architecture where the final three codons of MT-ND4L overlap with the initial codons of MT-ND4.

• Evolutionary implications: This conservation of overlap across species suggests functional significance, possibly in coordinating the expression of these two Complex I components.

While this specific overlap has been characterized in human mtDNA, similar overlapping arrangements likely exist in Metachirus nudicaudatus and should be verified through genomic analysis before designing experiments targeting either gene .

What techniques are recommended for studying MT-ND4L incorporation into Complex I?

Investigating MT-ND4L incorporation into Complex I requires specialized techniques due to its hydrophobic nature and importance in the complex assembly process:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE): This technique allows separation of intact respiratory complexes and subcomplexes while preserving native protein-protein interactions. It can reveal intermediates in Complex I assembly containing MT-ND4L.

• Pulse-chase labeling: Using radioactively labeled amino acids in combination with immunoprecipitation to track the incorporation of newly synthesized MT-ND4L into assembling Complex I.

• Import assays with isolated mitochondria: In vitro transcription and translation of MT-ND4L followed by import into isolated mitochondria can help study the integration process.

• Cryo-electron microscopy: For structural studies of MT-ND4L within the assembled complex, providing insights into its precise location and interactions.

• Proximity labeling with APEX2: Fusing APEX2 to MT-ND4L allows biotinylation of proteins in close proximity, identifying assembly partners during the incorporation process.

These techniques should be adapted to account for the specific properties of Metachirus nudicaudatus MT-ND4L, particularly its hydrophobicity and role in the transmembrane domain of Complex I .

What assays can be used to measure MT-ND4L functionality in experimental systems?

Several specialized assays can be employed to evaluate MT-ND4L functionality within the context of Complex I activity:

• NADH:ubiquinone oxidoreductase activity assay: This spectrophotometric assay measures the rate of NADH oxidation coupled to ubiquinone reduction, providing a direct assessment of Complex I function.

• Mitochondrial membrane potential measurements: Using fluorescent dyes like TMRM or JC-1 to assess whether MT-ND4L mutations or modifications affect the proton-pumping capacity of Complex I.

• Oxygen consumption rate (OCR): Measuring oxygen consumption using platforms like Seahorse XF Analyzer can assess the impact of MT-ND4L modifications on mitochondrial respiration.

• ROS production assays: Since Complex I dysfunction often results in increased reactive oxygen species production, measuring H₂O₂ or superoxide production can indicate functional alterations in MT-ND4L.

• Complex I assembly analysis: Using antibodies against various Complex I subunits in combination with BN-PAGE to determine if mutations in MT-ND4L affect the assembly or stability of the entire complex.

For accurate results, researchers should include appropriate controls, such as known inhibitors of Complex I (rotenone), and normalize measurements to mitochondrial content using citrate synthase activity or other established markers .

How can researchers optimize expression systems for recombinant MT-ND4L production?

Producing functional recombinant MT-ND4L presents significant challenges due to its hydrophobic nature and mitochondrial origin. Researchers can optimize expression using the following approaches:

• Expression system selection:

  • Bacterial systems (E. coli): Use specialized strains like C41(DE3) or C43(DE3) designed for membrane protein expression

  • Yeast systems: Pichia pastoris or Saccharomyces cerevisiae provide eukaryotic post-translational modifications

  • Cell-free expression systems: Allow direct incorporation into liposomes or nanodiscs

• Fusion protein strategies:

  • N-terminal fusions with highly soluble partners (MBP, SUMO, or Thioredoxin)

  • Addition of purification tags that can be later removed via protease cleavage sites

• Solubilization optimization:

  • Screen multiple detergents (DDM, LDAO, Fos-choline)

  • Test lipid nanodisc incorporation for maintaining native-like environment

• Expression conditions:

  • Reduce temperature to 16-18°C during induction

  • Use lower inducer concentrations for slower expression

  • Optimize media composition with glycerol supplementation

• Purification considerations:

  • Use two-step purification protocols

  • Include glycerol in all buffers (typically 10-20%)

  • Add lipids during purification to stabilize structure

These approaches can be tailored specifically for Metachirus nudicaudatus MT-ND4L based on its sequence characteristics and hydrophobicity profile .

How does MT-ND4L contribute to our understanding of mitochondrial disease mechanisms?

MT-ND4L research provides significant insights into mitochondrial disease mechanisms through several key contributions:

• Complex I dysfunction: MT-ND4L variants can disrupt Complex I assembly or function, leading to bioenergetic defects that characterize many mitochondrial disorders. As a core transmembrane component, its mutations can affect proton translocation and energy production.

• Alzheimer's Disease connection: Recent findings with the rs28709356 variant demonstrate a significant association between MT-ND4L and Alzheimer's Disease (p = 7.3 × 10^-5), supporting the mitochondrial cascade hypothesis of neurodegeneration. This provides a molecular link between mitochondrial dysfunction and cognitive decline.

• Reactive Oxygen Species (ROS) production: Alterations in MT-ND4L can increase electron leakage from Complex I, enhancing oxidative stress through excessive ROS generation—a common pathological mechanism in mitochondrial diseases.

• Evolutionary insights: Comparative studies of MT-ND4L across species, including Metachirus nudicaudatus, reveal conserved regions essential for function, helping identify pathogenic variants in human patients.

• Tissue-specific vulnerability: Research on MT-ND4L helps explain why certain tissues are preferentially affected in mitochondrial disorders, based on their reliance on oxidative phosphorylation and Complex I function.

This research contributes to developing more targeted therapeutic approaches for mitochondrial disorders by identifying specific functional domains and mechanisms disrupted by disease-causing mutations .

What is the significance of studying MT-ND4L in marsupials compared to placental mammals?

Studying MT-ND4L in marsupials like Metachirus nudicaudatus compared to placental mammals provides valuable evolutionary and functional insights:

Comparative analysis between the MT-ND4L of Metachirus nudicaudatus (Q5QS97) and placental mammals provides a window into the evolutionary processes shaping mitochondrial function and may identify novel functional elements not apparent from studying placental mammals alone .

How might MT-ND4L research inform our understanding of neurodegenerative disorders?

MT-ND4L research offers critical insights into neurodegenerative disorders through multiple mechanisms:

• Direct genetic association: The identification of the rare MT-ND4L variant rs28709356 (C>T) significantly associated with Alzheimer's Disease (p = 7.3 × 10^-5) provides direct evidence linking this gene to neurodegeneration. This finding supports mitochondrial dysfunction as a contributing factor to AD pathogenesis.

• Complex I deficiency in neurodegeneration: Neurons are particularly vulnerable to Complex I defects due to their high energy demands. MT-ND4L, as a core component of Complex I, may contribute to the selective neuronal vulnerability observed in conditions like Parkinson's disease and Alzheimer's disease.

• Bioenergetic failure mechanism: MT-ND4L dysfunction can impair ATP production through oxidative phosphorylation, triggering energy deficits that compromise neuronal functions including synaptic transmission, protein clearance, and calcium homeostasis.

• Oxidative stress pathways: Defects in MT-ND4L can increase ROS production from Complex I, promoting oxidative damage to proteins, lipids, and DNA—all recognized contributors to neurodegenerative processes.

• Potential therapeutic targets: Understanding MT-ND4L structure and function across species, including Metachirus nudicaudatus, may identify conserved regions that could serve as targets for developing therapies aimed at enhancing mitochondrial function in neurodegenerative disorders.

The gene-based association of MT-ND4L with Alzheimer's Disease (p = 6.71 × 10^-5) underscores the importance of further research into mitochondrial genes as contributors to neurodegenerative pathology and potential therapeutic targets .

What are the implications of using base editing approaches for MT-ND4L functional studies?

The development of base editing techniques specifically for mitochondrial genes like MT-ND4L represents a significant methodological advancement with several important implications:

• Heteroplasmy control: Base editing approaches allow researchers to generate cells with controlled levels of MT-ND4L mutations, enabling precise studies of threshold effects in mitochondrial dysfunction.

• Pathogenic variant modeling: The ability to convert specific codons to stop codons (as demonstrated with the Val90-Gln91 region in mouse MT-ND4L) permits the creation of models mimicking disease-associated truncations.

• Tissue-specific phenotypes: Combined with tissue-specific expression systems, mitochondrial base editing can facilitate investigation of how MT-ND4L disruption affects different cell types, addressing the question of why certain tissues are preferentially affected in mitochondrial disorders.

• Therapeutic potential: While currently used for research purposes, these techniques may eventually be adapted for correcting pathogenic MT-ND4L variants in patients with mitochondrial disorders.

• Evolutionary studies: By introducing equivalent mutations across different species' MT-ND4L genes, researchers can compare phenotypic consequences and infer functional conservation.

The sequential transfection protocol demonstrated for mouse MT-ND4L editing, achieving effectively homoplasmic cells with premature stop codons, provides a robust methodological framework that can be adapted for studying Metachirus nudicaudatus MT-ND4L function and dysfunction .

How does MT-ND4L sequence analysis contribute to phylogenetic studies of marsupials?

MT-ND4L sequence analysis provides valuable data for phylogenetic studies of marsupials, offering several distinct advantages:

• Mitochondrial inheritance patterns: The maternal inheritance of mitochondrial genes like MT-ND4L allows tracking of matrilineal evolutionary history, providing complementary information to nuclear gene phylogenies.

• Evolutionary rate characteristics: MT-ND4L exhibits a relatively high evolutionary rate compared to many nuclear genes, making it useful for resolving relationships among closely related marsupial species.

• Conserved length with variable sequence: The consistent 98-amino acid length of MT-ND4L across diverse mammals, coupled with sequence variations, provides informative characters for phylogenetic analysis while maintaining structural constraints.

• Comparative data potential: Comparing Metachirus nudicaudatus MT-ND4L (Q5QS97) with other marsupials can reveal didelphid-specific adaptations and evolutionary patterns within this diverse marsupial family.

• Complementary marker to traditional loci: MT-ND4L can serve as an additional marker to corroborate or challenge phylogenetic hypotheses based on other mitochondrial genes (like cytochrome b) or nuclear loci.

Sequence analysis of Metachirus nudicaudatus MT-ND4L contributes to a more comprehensive understanding of marsupial evolutionary relationships and can help resolve phylogenetic questions within Didelphidae and broader marsupial taxa .

What are the key resources available for MT-ND4L research?

Researchers studying Metachirus nudicaudatus MT-ND4L have access to several key resources:

• Protein databases:

  • UniProt entry Q5QS97 containing sequence information for Metachirus nudicaudatus MT-ND4L

  • Comparative data from related species (e.g., Q5Y4Q2 for Bos mutus grunniens)

• Genetic resources:

  • Mitochondrial genome sequences for Metachirus nudicaudatus

  • Orthologous sequences across 293 species (as indicated in Ensembl for mouse mt-Nd4l)

• Experimental reagents:

  • Recombinant protein (50 μg quantities available, with other quantities by inquiry)

  • Storage and handling protocols specific to this protein

• Methodological resources:

  • Base editing protocols for mitochondrial genes, adaptable to MT-ND4L studies

  • Complex I functional assay methodologies

• Disease association data:

  • Information on MT-ND4L variants associated with Alzheimer's Disease

  • Methodologies for investigating pathogenic variants