Recombinant Galemys pyrenaicus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its role in cellular metabolism?

NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L) is a protein subunit of mitochondrial Complex I (NADH dehydrogenase) in the respiratory chain with EC number 1.6.5.3 . This inner mitochondrial membrane protein plays a critical role in the electron transport chain, facilitating the transfer of electrons from NADH to ubiquinone, which is essential for cellular energy production through oxidative phosphorylation. The protein comprises 98 amino acids in Galemys pyrenaicus, similar to the protein length seen in other species such as Microtus . MT-ND4L functions within the membrane arm of Complex I, contributing to proton pumping across the inner mitochondrial membrane, which drives ATP synthesis. Research suggests that MT-ND4L interacts closely with other Complex I subunits, including ND6 and NDUFS1, as demonstrated in protein-protein interaction studies .

What are the structural and genetic characteristics of MT-ND4L in Galemys pyrenaicus?

MT-ND4L in Galemys pyrenaicus is encoded by the mitochondrial genome (MT-ND4L gene) and consists of 98 amino acids with the sequence: MSLVYVNIMIAFSVSFLLGLLMFRSHLMSLLCLEGMMLSLFILIGTILILNFHFTLASMAPIIMLVFAACEAVGLSLLVMVSNTYGVDYVQNLNLLQC . Comparative studies with other mammalian species show that ND4L gene is typically about 297 base pairs in length, utilizing ATG as a start codon and TAA as a stop codon . The protein is highly hydrophobic with multiple transmembrane domains, consistent with its function as an integral membrane protein. In the context of the mitochondrial genome organization, MT-ND4L is part of a conserved gene arrangement found across mammalian species. The A+T content in related species ranges from approximately 55.89% to 59.60%, which is characteristic of mitochondrial genes . This protein exhibits significant sequence conservation across evolutionarily related species, particularly in regions critical for its functional integration within Complex I.

What expression systems are most effective for producing recombinant MT-ND4L?

For effective production of recombinant Galemys pyrenaicus MT-ND4L, a combination of optimization strategies is required due to its hydrophobic nature and mitochondrial origin. The most successful expression systems employ specialized vectors containing mitochondrial targeting sequences and appropriate membrane protein expression elements. Bacterial expression systems using E. coli strains specifically designed for membrane protein expression (such as C41(DE3) or C43(DE3)) can be effective when combined with fusion tags that enhance solubility.

Alternatively, eukaryotic systems including yeast (P. pastoris) or insect cell (Sf9, Sf21) expression systems may provide better folding environments for maintaining protein structure. Based on research with similar mitochondrial proteins, codon optimization for the expression host is crucial, as is the inclusion of appropriate purification tags (His-tag or other affinity tags) that do not interfere with protein folding . Additionally, incorporating the 3'UTR signals from nuclear genes encoding mitochondrial proteins (like COX10) has been shown to improve expression and mitochondrial targeting of recombinant mitochondrial proteins, as demonstrated in related research .

How can recombinant MT-ND4L be used in phylogenetic studies of Talpidae and Eulipotyphla?

Recombinant MT-ND4L can serve as a valuable tool in phylogenetic studies of Talpidae (the family including Galemys pyrenaicus) and broader Eulipotyphla taxonomic investigations through several methodological approaches. Researchers can utilize the recombinant protein to develop specific antibodies that enable comparative immunohistochemical studies across species, revealing evolutionary patterns in protein localization and interaction networks.

A multigene approach incorporating MT-ND4L sequence data alongside other mitochondrial and nuclear genes has proven effective in resolving phylogenetic relationships within these taxonomic groups . Specifically, complete mitochondrial genome sequencing that includes MT-ND4L has helped clarify the phylogenetic position of Galemys pyrenaicus within Talpidae and Eulipotyphla . Recombinant protein can also be used in functional comparative studies to assess enzymatic activity differences between species, potentially revealing signatures of adaptive evolution.

The methodological framework should include:

• PCR amplification using primers such as Gale CytbF (5′-CAAACATCTCATCATGATGRAA-3′) and Gale Cytb2R (5′-TGTTTTCTATAATGCTTGCTAGTGG-3′)

• Sequencing of the amplified products

• Comparative sequence analysis across species

• Integration of MT-ND4L data with other genetic markers for comprehensive phylogenetic reconstruction

This approach has successfully contributed to understanding the evolutionary relationships of the Pyrenean desman within the mammalian tree of life and can be applied to other taxonomic questions .

What experimental protocols are recommended for studying MT-ND4L protein-protein interactions within Complex I?

For investigating MT-ND4L protein-protein interactions within Complex I, a multi-methodological approach is recommended. Based on successful research with related mitochondrial proteins, the following protocol framework is advised:

• Proximity Ligation Assay (PLA): This technique can effectively detect in situ protein-protein interactions between MT-ND4L and other Complex I subunits. Research has demonstrated that PLA successfully identified interactions between ND4-HA1 and other subunits including ND6 and NDUFS1, appearing as bright fluorescent dots in tissue sections . The protocol should include appropriate antibody pairs (anti-MT-ND4L and antibodies against potential interaction partners).

• Co-immunoprecipitation with tagged recombinant protein: Using recombinant MT-ND4L with an affinity tag (such as HA1) allows pull-down experiments to identify interaction partners. This approach has shown that ND4 interacts with ND6 and NDUFS1, while showing minimal interaction with proteins like VDAC or OPA1 .

• Blue Native PAGE followed by Western blotting: This technique preserves protein complexes and can demonstrate the incorporation of recombinant MT-ND4L into Complex I.

• Immunofluorescence co-localization studies: Using confocal microscopy with antibodies against MT-ND4L and other Complex I subunits can reveal spatial relationships within cellular contexts. The pattern typically appears as punctate fluorescent dots excluded from the nuclei, with yellow-orange pixels in merged images indicating co-localization .

For optimal results, these techniques should be applied in complementary fashion, as each provides different insights into the interaction network of MT-ND4L within the respiratory complex.

How can MT-ND4L mutations be analyzed for their impact on mitochondrial function?

Analysis of MT-ND4L mutations and their impact on mitochondrial function requires a systematic approach combining genetic, biochemical, and cellular techniques. The recommended methodological framework includes:

• Generation of mutation models: Either through site-directed mutagenesis of recombinant MT-ND4L or through techniques like allotopic expression, where nuclear versions of the mitochondrial gene (containing the desired mutations) are introduced into cells or animal models. This approach has been successfully employed for related proteins like ND4 .

• Complex I activity assays: Spectrophotometric measurements of NADH oxidation or ubiquinone reduction rates in isolated mitochondria or tissue homogenates from models expressing mutant MT-ND4L. Compare results with wild-type controls to quantify the degree of dysfunction.

• Oxygen consumption analysis: Using respirometry techniques to measure oxygen consumption rates in intact cells or isolated mitochondria expressing mutant MT-ND4L.

• ROS production assessment: Employing fluorescent probes specific for reactive oxygen species to determine if MT-ND4L mutations increase oxidative stress, a common consequence of respiratory chain dysfunction.

• Mitochondrial membrane potential measurement: Using potential-sensitive dyes to assess if mutations affect the proton-pumping function of Complex I.

• Cell viability and apoptosis assays: To determine if mutations in MT-ND4L lead to decreased cell survival or increased apoptosis rates.

This integrated approach has been validated in studies of other mitochondrial gene mutations, including those in ND4, where researchers successfully demonstrated that mutations led to respiratory chain dysfunction and subsequently to retinal ganglion cell degeneration in models of Leber hereditary optic neuropathy .

What is the potential of MT-ND4L as a therapeutic target for mitochondrial disorders?

MT-ND4L represents a potentially significant therapeutic target for mitochondrial disorders, particularly those involving Complex I dysfunction. The therapeutic potential can be explored through several strategic approaches:

• Gene therapy through optimized allotopic expression: This approach involves creating a nuclear version of the MT-ND4L gene with appropriate targeting sequences to ensure the protein is imported into mitochondria. Research with the related ND4 gene has demonstrated that optimized allotopic expression can prevent retinal ganglion cell degeneration and preserve complex I function in optic nerves in models of Leber hereditary optic neuropathy . The optimization includes using the 3'UTR of the COX10 gene, which facilitates mRNA localization to the mitochondrial surface where translation and transport can be coupled .

• Structure-based drug design: Advanced AI-driven conformational ensemble generation has been applied to MT-ND4L to identify binding pockets that could be targeted with small molecules. This approach integrates AI algorithms to predict alternative functional states of the protein and identify orthosteric, allosteric, hidden, and cryptic binding pockets .

• Peptide-based approaches: Developing peptides that can stabilize or correct the folding of mutant MT-ND4L or its interactions with other Complex I subunits.

Research demonstrates that targeting mitochondrial proteins like MT-ND4L has therapeutic potential, as interventions that restore even partial Complex I function can significantly ameliorate disease phenotypes in mitochondrial disorders . The therapeutic strategy would need to be tailored to the specific mutation and disease context, with consideration of appropriate delivery methods to reach the affected tissues.

How can recombinant MT-ND4L be used in drug discovery pipelines for mitochondrial disease treatments?

Recombinant MT-ND4L can be strategically integrated into drug discovery pipelines for mitochondrial disease treatments through a structured approach:

• High-throughput screening platforms: Developing assays using recombinant MT-ND4L incorporated into artificial membrane systems or reconstituted Complex I to screen compound libraries for molecules that stabilize or enhance function.

• AI-driven molecular design: Utilizing recombinant protein for structural characterization that feeds into AI algorithms for conformational ensemble generation. This approach has been employed to explore the conformational space of MT-ND4L and identify representative structures that capture the protein's full dynamic behavior .

• Binding pocket identification and characterization: Employing AI-based pocket prediction modules that integrate literature search data with structure-aware ensemble-based detection algorithms to discover orthosteric, allosteric, hidden, and cryptic binding pockets on the protein's surface .

• Target validation studies: Using recombinant protein for binding assays, thermal shift assays, and other biophysical techniques to validate potential drug candidates identified through virtual screening.

• Cell-based functional assays: Developing assay systems where the impact of compounds on MT-ND4L function can be assessed in cellular contexts, measuring endpoints such as Complex I activity, ATP production, and cell viability.

This integrated approach leverages both computational and experimental methods, with recombinant MT-ND4L serving as a critical tool throughout the process. The methodology has proven effective in identifying prospective binding sites and potential therapeutic compounds for other mitochondrial proteins .

What techniques are recommended for studying the incorporation of recombinant MT-ND4L into the mitochondrial respiratory complex?

Studying the incorporation of recombinant MT-ND4L into the mitochondrial respiratory complex presents several technical challenges that can be addressed through the following methodological approaches:

• Immunofluorescence co-localization analysis: Using antibodies against both the recombinant MT-ND4L (typically with an epitope tag such as HA1) and endogenous mitochondrial proteins like ND6. Successful incorporation appears as punctate fluorescent dots in the cytoplasm with significant co-localization between the tagged protein and other Complex I subunits . This technique has demonstrated that human ND4 protein can be efficiently imported inside mitochondria and assembled in respiratory chain Complex I .

• Blue Native PAGE and Western blotting: This technique preserves protein complexes during electrophoresis and can demonstrate whether recombinant MT-ND4L is assembled into the ~1 MDa Complex I. Sequential immunoblotting with antibodies against the recombinant protein and known Complex I subunits can confirm incorporation.

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions in situ with high sensitivity and specificity. PLA has successfully shown that ND4-HA1 interacts with ND6 and NDUFS1, appearing as bright red dots in tissue sections, while showing minimal interaction with non-Complex I mitochondrial proteins like VDAC or OPA1 .

• Complex I activity assays: Functional incorporation can be assessed by measuring Complex I activity in systems expressing recombinant MT-ND4L compared to controls. Restoration of Complex I function in deficient models provides strong evidence for successful incorporation.

• Submitochondrial fractionation: Isolating inner membrane complexes and analyzing the presence of recombinant MT-ND4L through mass spectrometry or immunoblotting.

These techniques, when used in combination, provide robust evidence for the mitochondrial localization and functional incorporation of recombinant MT-ND4L into the respiratory chain complex.

What are the key considerations for analyzing MT-ND4L evolutionary conservation across species?

Analysis of MT-ND4L evolutionary conservation across species requires attention to several methodological considerations:

• Sequence alignment and comparison methodology: Employing appropriate alignment algorithms that account for the highly hydrophobic nature of MT-ND4L. Research on Microtus species showed 82-83% identity between species for the ND4L gene, highlighting the importance of proper alignment techniques . Multiple sequence alignment tools such as MUSCLE or CLUSTAL should be optimized for membrane proteins.

• Codon usage analysis: MT-ND4L exhibits specific codon usage patterns that may vary across species but retain functional constraints. Analysis of codon usage can provide insights into evolutionary selection pressures .

• A+T content assessment: The A+T content of MT-ND4L varies across species (55.89-59.60% in Microtus species) and can be informative for evolutionary studies . Systematic analysis of nucleotide composition across taxa should be incorporated into conservation studies.

• Start/stop codon analysis: MT-ND4L typically uses ATG as a start codon and TAA as a stop codon, but variations exist across species . Documentation of these variations can provide insights into evolutionary mechanisms.

• Phylogenetic methodology selection: Using appropriate phylogenetic reconstruction methods (Maximum Likelihood, Bayesian Inference) that account for the characteristics of mitochondrial sequences. A multigene approach incorporating MT-ND4L sequence data alongside other mitochondrial and nuclear genes has proven effective in resolving phylogenetic relationships .

• Functional domain conservation: Analyzing conservation patterns in the context of functional domains and protein-protein interaction sites within Complex I.

• Selection pressure analysis: Calculating dN/dS ratios to identify regions under purifying or positive selection.

The comprehensive approach to evolutionary conservation analysis should integrate these methods to provide insights into both sequence and functional conservation of MT-ND4L across diverse taxonomic groups.

How should researchers interpret comparative data on MT-ND4L sequence variation in relation to species phylogeny?

When interpreting comparative data on MT-ND4L sequence variation in relation to species phylogeny, researchers should employ a structured analytical framework:

• Contextualize sequence identity metrics: Sequence identity percentages should be interpreted within the broader context of mitochondrial genome evolution. For example, in Microtus species, ND4L shows 82-83% identity between species, which is lower than some other mitochondrial genes like CytB (87-88% identity) . This comparative approach helps identify whether MT-ND4L is evolving at a typical rate for mitochondrial genes in the lineage of interest.

• Integrate with multi-gene analyses: MT-ND4L sequence data should be analyzed alongside other mitochondrial and nuclear genes for comprehensive phylogenetic reconstruction. This approach has been successfully used to clarify the phylogenetic position of Galemys pyrenaicus within Talpidae and Eulipotyphla .

• Consider functional constraints: Interpret sequence conservation in light of the protein's functional domains and constraints. Regions involved in protein-protein interactions or essential for catalytic function typically show higher conservation.

• Analyze codon position variability: First, second, and third codon positions evolve at different rates, with third positions typically showing higher variability. Analysis of position-specific variation can provide insights into selection pressures.

• Evaluate phylogenetic signal: Test for phylogenetic signal using metrics like the consistency index or retention index to determine if MT-ND4L variation reflects true evolutionary relationships or homoplasy.

• Consider rate heterogeneity: Account for site-specific and lineage-specific rate variation in analyses, as mitochondrial genes can show heterogeneous evolutionary rates across sites and lineages.

• Correlate with ecological adaptations: Where possible, interpret sequence variations in the context of species-specific ecological adaptations that might influence mitochondrial function.

This comprehensive interpretative framework ensures that MT-ND4L sequence data contributes meaningfully to phylogenetic studies while accounting for the complexities of mitochondrial genome evolution.

What statistical approaches are most appropriate for analyzing MT-ND4L sequence data in phylogenetic studies?

For robust analysis of MT-ND4L sequence data in phylogenetic studies, researchers should employ the following statistical approaches:

• Model selection and testing: Use statistical criteria such as Akaike Information Criterion (AIC), Bayesian Information Criterion (BIC), or hierarchical likelihood ratio tests to select the most appropriate model of nucleotide or amino acid substitution for MT-ND4L. This is critical as inappropriate model selection can lead to phylogenetic artifacts.

• Bayesian phylogenetic inference: Implement Bayesian methods using programs like MrBayes or BEAST, which allow for complex evolutionary models and provide measures of parameter uncertainty through posterior probability distributions. This approach has been successfully applied in phylogenetic studies incorporating mitochondrial genome data from Galemys pyrenaicus .

• Maximum Likelihood analysis: Employ ML methods with appropriate bootstrapping (typically 1000 replicates) to assess the statistical support for nodes in the phylogenetic tree.

• Partition-specific analysis: Analyze different codon positions or functional domains separately, as they may evolve under different constraints. This partitioned analysis approach can improve phylogenetic resolution.

• Tests for selection: Implement tests for positive or negative selection using dN/dS ratios calculated with methods such as PAML, which can identify specific sites or lineages under selection.

• Molecular clock tests: Apply likelihood ratio tests or relative rate tests to examine whether MT-ND4L evolves in a clock-like manner or shows rate variation across lineages.

• Congruence testing: Statistically test for congruence between phylogenetic signals from MT-ND4L and other genes using methods like the incongruence length difference test or Shimodaira-Hasegawa test.

• Ancestral sequence reconstruction: Employ maximum likelihood or Bayesian methods to reconstruct ancestral MT-ND4L sequences at key nodes in the phylogeny, providing insights into the evolution of the protein.

These statistical approaches, when applied rigorously and in combination, ensure that phylogenetic inferences based on MT-ND4L sequence data are robust and properly account for the complexities of molecular evolution.

How can single-molecule techniques be applied to study MT-ND4L function and dynamics?

Single-molecule techniques offer unprecedented insights into the function and dynamics of membrane proteins like MT-ND4L. Researchers can implement these advanced methodologies through the following approaches:

• Single-molecule FRET (smFRET): By strategically placing fluorophore pairs on recombinant MT-ND4L and interacting Complex I subunits, researchers can monitor conformational changes during the catalytic cycle in real-time. This requires careful selection of labeling sites that don't interfere with protein function and optimization of reconstitution into membrane mimetics such as nanodiscs or liposomes.

• Atomic Force Microscopy (AFM): High-resolution AFM can be used to visualize the topography of MT-ND4L within the Complex I structure and measure interaction forces between MT-ND4L and other subunits or substrates. This approach has been successfully applied to other membrane protein complexes.

• Single-molecule electrophysiology: Patch-clamp techniques adapted for reconstituted systems can potentially measure ion or proton translocation associated with MT-ND4L function within Complex I.

• Optical tweezers combined with fluorescence microscopy: This approach can be used to measure mechanical forces during protein folding or complex assembly, providing insights into the energetics of MT-ND4L incorporation into Complex I.

• Single-particle tracking: Using quantum dots or other bright, photostable fluorophores conjugated to recombinant MT-ND4L to track its movement and distribution within mitochondrial membranes in live cells.

• Super-resolution microscopy: Techniques such as STORM or PALM can overcome the diffraction limit to visualize the nanoscale organization of MT-ND4L within the mitochondrial membrane with precision approaching 20 nm.

These cutting-edge approaches require significant technical expertise and optimization but offer the potential to reveal fundamental aspects of MT-ND4L function that cannot be observed with ensemble methods.

What are the current hypotheses regarding the role of MT-ND4L in mitochondrial disease pathogenesis?

Current hypotheses regarding the role of MT-ND4L in mitochondrial disease pathogenesis center around several mechanistic frameworks that can guide research:

• Complex I assembly disruption hypothesis: Mutations in MT-ND4L may prevent proper assembly of Complex I, leading to reduced activity and energy production. This is supported by evidence from studies of other Complex I subunits like ND4, where mutations affect complex assembly and stability .

• Proton pumping efficiency hypothesis: Given its location in the membrane arm of Complex I, MT-ND4L may play a role in proton translocation. Mutations could disrupt the efficiency of this process, uncoupling electron transport from ATP synthesis and reducing energy production while potentially increasing reactive oxygen species (ROS) generation.

• Protein-protein interaction disruption hypothesis: MT-ND4L interacts with multiple Complex I subunits including ND6 and NDUFS1 . Mutations may disrupt these interactions, compromising complex stability and function. This is supported by proximity ligation assay studies showing specific interactions between Complex I subunits.

• Tissue-specific vulnerability hypothesis: Certain tissues with high energy demands, particularly neural tissues, may be especially vulnerable to MT-ND4L dysfunction. This parallels the tissue-specific effects seen with mutations in other mitochondrial genes like ND4, which cause Leber hereditary optic neuropathy affecting retinal ganglion cells .

• ROS production hypothesis: Dysfunction in MT-ND4L may increase electron leakage from Complex I, leading to elevated ROS production and oxidative damage. This oxidative stress hypothesis is consistent with findings from other Complex I deficiencies.

• Compensatory mechanisms hypothesis: The severity of phenotypes caused by MT-ND4L mutations may depend on cellular capacity to compensate through upregulation of alternative energy production pathways or mitochondrial biogenesis.

Research approaches testing these hypotheses should include functional studies in relevant cell and animal models, potentially using gene therapy approaches similar to those developed for ND4-related disorders .