Recombinant Zalophus californianus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

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

MT-ND4L is a gene found in the mitochondrial genome that codes for the NADH-ubiquinone oxidoreductase chain 4L protein. This protein serves as a critical subunit of NADH dehydrogenase (ubiquinone), also known as Complex I, which is located in the mitochondrial inner membrane. Complex I represents the largest of the five complexes in the electron transport chain responsible for cellular respiration . The ND4L protein specifically contributes to the core functionality of Complex I by participating in proton translocation across the mitochondrial membrane, which is essential for establishing the proton gradient that drives ATP synthesis. Research has established that ND4L is one of the most hydrophobic subunits of Complex I and forms part of the core transmembrane region, highlighting its importance in maintaining the structural integrity of this respiratory complex .

What is the genomic structure and location of MT-ND4L in mitochondrial DNA?

In humans, the MT-ND4L gene is precisely located within the mitochondrial DNA from base pair 10,469 to 10,765. The gene encodes a relatively small protein of approximately 11 kDa, composed of 98 amino acids . MT-ND4L is one of seven mitochondrial genes encoding subunits of Complex I, alongside MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, and MT-ND6. An interesting characteristic of the human MT-ND4L gene is its unusual 7-nucleotide overlap with the MT-ND4 gene. Specifically, the last three codons of MT-ND4L (5'-CAA TGC TAA-3' coding for Gln, Cys, and Stop) overlap with the first three codons of MT-ND4 (5'-ATG CTA AAA-3' coding for Met-Leu-Lys). With respect to the MT-ND4L reading frame (+1), the MT-ND4 gene starts in the +3 reading frame . This overlapping gene arrangement represents an efficient use of the compact mitochondrial genome and has important implications for the coordinated expression of these functionally related proteins.

How is MT-ND4L conserved across species, particularly in marine mammals?

The conservation of MT-ND4L across different species, especially marine mammals like Zalophus californianus (California sea lion), reflects its evolutionary importance. Genetic analysis of the Japanese sea lion (Zalophus californianus japonicus), which is now practically extinct, has provided valuable insights into the evolutionary patterns of MT-ND4L in these species . Molecular phylogenetic analysis using the neighbor-joining method has shown that sequences from Japanese sea lions form a distinct cluster with high bootstrap values, positioned closest to the California sea lion cluster. The average nucleotide substitution between Japanese and California sea lions is approximately 7.02%, suggesting their divergence occurred approximately 2.2 million years ago in the late Pliocene Epoch . This evolutionary conservation underscores the functional importance of MT-ND4L in cellular energy metabolism across diverse mammalian lineages.

What techniques are most effective for the expression and purification of recombinant Zalophus californianus MT-ND4L?

When working with recombinant Zalophus californianus MT-ND4L, researchers should consider the highly hydrophobic nature of this protein, which presents unique challenges for expression and purification. Based on available research protocols, the following methodological approach is recommended:

• Expression System Selection: Due to the mitochondrial origin of MT-ND4L, specialized expression systems that can accommodate membrane proteins are preferred. Bacterial systems (E. coli) with modified strains designed for membrane protein expression have shown success.

• Fusion Tag Strategy: Incorporating solubility-enhancing tags such as SUMO or MBP can improve expression yields. For the recombinant Zalophus californianus MT-ND4L, tag types are typically determined during the production process to optimize for protein stability and functionality .

• Purification Protocol: A multi-step purification process involving detergent solubilization followed by affinity chromatography is commonly employed. The recombinant protein is typically stored in a Tris-based buffer with 50% glycerol to maintain stability .

• Quality Control: Verification of protein identity is essential, with confirmation against the known amino acid sequence: MSMMYFNIFMAFTVSLVGLLMYRSHLMSSLLCLEGMMLSLFVMMSVTILNNHFTLASMAPIILLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC, corresponding to positions 1-98 of the full-length protein .

For optimal storage stability, aliquots should be maintained at -20°C for short-term storage, while extended storage requires conservation at -80°C. Repeated freeze-thaw cycles should be avoided to preserve protein integrity .

How can researchers effectively study the structural dynamics of MT-ND4L using molecular simulation approaches?

Molecular dynamics (MD) simulations provide valuable insights into the structural dynamics of MT-ND4L and its role in proton translocation. A methodologically sound approach involves:

• Model Preparation: Begin by obtaining or constructing a high-quality structural model of MT-ND4L. When studying Zalophus californianus MT-ND4L, researchers often need to employ homology modeling based on available structures from related species or human Complex I components .

• Simulation Parameters: Conduct simulations spanning at least 100 ns to capture meaningful conformational changes. Established molecular dynamics packages such as AMBER18 have proven effective for simulating membrane proteins like MT-ND4L .

• Analysis of Proton Pathways: Focus analysis on identifying water channels and conserved amino acids involved in proton translocation. Research has shown that MT-ND4L contributes to the fourth proton channel in respiratory Complex I, making this a critical area of investigation .

• Mutation Impact Assessment: Compare native and mutant models to evaluate how specific amino acid changes affect proton translocation mechanisms. For example, studies on human MT-ND4L have demonstrated that certain mutations (such as M47T from T10609C mutation and C69W from C10676G mutation) can interrupt the proton translocation pathway by forming hydrogen bonds between specific residues (e.g., Glu34 and Tyr157) .

• Visualization and Analysis: Employ specialized visualization tools like VMD (Visual Molecular Dynamics) to analyze water molecule behavior, hydrogen bonding patterns, and conformational changes that may impact protein function .

This methodology has successfully demonstrated how mutations in MT-ND4L can restrict the passage of water molecules through the transmembrane region, potentially affecting mitochondrial function and contributing to conditions such as diabetes mellitus and cataracts .

What associations exist between MT-ND4L variants and metabolomic profiles?

Genome-wide association studies (GWAS) with metabolomics have revealed significant associations between MT-ND4L variants and specific metabolite ratios. In a study of 1163 individuals with sequenced mitochondria covering 9172 mtSNVs, several significant associations were identified involving MT-ND4L variants . The research methodology involved:

• Linear regression analysis of 151 metabolic traits

• Application of stringent significance criteria (P-value < 1.257545 × 10⁻⁵ after M_eff correction)

• Verification of metabolite ratio P-gain values (>151)

The study identified 404 mtSNVs with genome-wide significant metabolite ratio associations. Notably, a significant proportion (15%) of the most statistically significant mtSNVs were located in MT-ND4L, predominantly associated with glycerophospholipid class metabolites .

Key findings related to MT-ND4L variants include:

These associations between MT-ND4L variants and metabolite ratios suggest that mitochondrial genetic variations can significantly influence metabolic pathways, particularly those involving phospholipid metabolism . This offers potential biomarkers for mitochondrial function assessment and could have implications for understanding metabolic diseases.

How do mutations in MT-ND4L impact mitochondrial respiratory complex function?

Mutations in MT-ND4L can significantly impact the function of mitochondrial respiratory Complex I through several mechanisms:

• Disruption of Proton Translocation: Molecular dynamics simulations have demonstrated that mutations in MT-ND4L, such as M47T (resulting from T10609C) and C69W (resulting from C10676G), can interrupt the proton translocation pathway. This occurs through the formation of abnormal hydrogen bonds between amino acid residues like Glu34 and Tyr157 .

• Altered Water Channel Function: Studies have shown that these mutations restrict the passage of water molecules through the transmembrane region, which is critical for proton movement. The native MT-ND4L model demonstrates proton translocation pathways similar to those observed in Complex I from various organisms, while mutant forms show distinctive interruptions in these pathways .

• Energy Production Impairment: By interfering with proton translocation, MT-ND4L mutations can reduce the efficiency of the proton gradient generation across the inner mitochondrial membrane, ultimately affecting ATP synthesis and cellular energy production.

• Association with Metabolic Disorders: Variants in MT-ND4L have been linked to increased BMI in adults and have shown associations with specific metabolite profiles, particularly those involving glycerophospholipids . Computational studies suggest that certain mutations could serve as potential genetic biomarkers for conditions such as Type 2 diabetes mellitus and cataracts .

These findings underscore the importance of MT-ND4L in maintaining proper respiratory chain function and highlight how specific mutations can lead to functional impairments with potential disease implications.

How can recombinant Zalophus californianus MT-ND4L be utilized in comparative evolutionary studies?

Recombinant Zalophus californianus MT-ND4L offers valuable opportunities for comparative evolutionary studies across marine mammals and other species. A methodological framework for such research includes:

• Ancient DNA Analysis: The extraction and sequencing of MT-ND4L from ancient specimens, as demonstrated with the Japanese sea lion (Zalophus californianus japonicus), can provide insights into evolutionary relationships. This approach has successfully revealed that Japanese and California sea lions diverged approximately 2.2 million years ago .

• Phylogenetic Analysis: Constructing molecular phylogenetic trees using neighbor-joining methods can help establish relationships between different species and subspecies. This technique has supported the classification of Japanese sea lions as a distinct species while also confirming their close relationship to California sea lions .

• Structural Conservation Assessment: Comparative analysis of recombinant MT-ND4L proteins from different species can reveal conserved functional domains and species-specific adaptations. This approach helps identify critical regions that have been maintained throughout evolution versus those that have undergone adaptive changes.

• Functional Comparative Studies: In vitro assays comparing the biochemical properties of recombinant MT-ND4L from different species can highlight functional adaptations. These studies can examine differences in:

  • Protein stability under various conditions

  • Interaction with other Complex I subunits

  • Efficiency of proton translocation

• Environmental Adaptation Analysis: Comparing MT-ND4L sequences and functions across species adapted to different environments (terrestrial vs. marine mammals) can reveal molecular adaptations to specific ecological niches.

These comparative approaches provide insights into both evolutionary relationships and the functional significance of MT-ND4L conservation or divergence across species, contributing to our understanding of mitochondrial evolution and adaptation.

What are the implications of MT-ND4L variants for human disease research?

Research on MT-ND4L variants has revealed significant implications for human disease studies:

• Association with Leber's Hereditary Optic Neuropathy (LHON): Variants of human MT-ND4L have been associated with LHON, a maternally inherited form of vision loss that primarily affects central vision . Understanding the molecular mechanisms of MT-ND4L dysfunction can provide insights into the pathophysiology of this condition.

• Metabolic Disorder Biomarkers: Genome-wide association studies have identified significant associations between MT-ND4L variants and specific metabolite ratios, particularly those involving glycerophospholipids . These associations may serve as potential biomarkers for metabolic disorders and could inform diagnostic approaches.

• Diabetes and Cataract Research: Molecular dynamics simulations have demonstrated how specific mutations in MT-ND4L (M47T and C69W) can disrupt proton translocation pathways. These findings have potential applications in developing computational assays for validating genetic biomarkers for Type 2 diabetes mellitus and cataracts .

• Obesity Research: Variants of human MT-ND4L have been associated with increased BMI in adults , suggesting a potential role in metabolic regulation and energy homeostasis. This connection provides a research avenue for investigating mitochondrial contributions to metabolic syndrome and obesity.

• Therapeutic Target Development: Understanding the structural and functional implications of MT-ND4L variants opens possibilities for developing targeted therapeutic approaches. Computational models that accurately simulate the effects of mutations on protein function can guide the design of compounds that might restore normal function or mitigate the effects of pathogenic variants.

These disease associations highlight the importance of continued research on MT-ND4L and underscore its potential as a target for diagnostic and therapeutic development across multiple human diseases with mitochondrial components.

What are the primary challenges in working with recombinant MT-ND4L and how can they be addressed?

Working with recombinant MT-ND4L presents several technical challenges due to its biochemical properties and mitochondrial origin. Here are the major challenges and methodological solutions:

• High Hydrophobicity

  • Challenge: MT-ND4L is one of the most hydrophobic subunits of Complex I , making expression and solubilization difficult.

  • Solution: Employ specialized membrane protein expression systems and optimize buffer conditions with appropriate detergents. Storage in 50% glycerol can help maintain protein stability .

• Proper Folding and Orientation

  • Challenge: As a mitochondrial membrane protein, MT-ND4L requires specific conditions to achieve proper folding.

  • Solution: Consider co-expression with chaperone proteins or other Complex I subunits, particularly those that interact directly with MT-ND4L in the native complex.

• Functional Assessment

  • Challenge: Testing the functionality of isolated MT-ND4L outside its normal complex is difficult.

  • Solution: Develop reconstituted systems that incorporate MT-ND4L into artificial membranes or nanodiscs to assess proton translocation activity. Molecular dynamics simulations can also provide insights into functional properties .

• Species-Specific Optimizations

  • Challenge: Working with Zalophus californianus MT-ND4L requires species-specific optimizations.

  • Solution: Start with established protocols for mammalian MT-ND4L and adjust based on the specific amino acid sequence (MSMMYFNIFMAFTVSLVGLLMYRSHLMSSLLCLEGMMLSLFVMMSVTILNNHFTLASMAPIILLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC) .

• Storage Stability

  • Challenge: Maintaining the stability of purified recombinant MT-ND4L during storage.

  • Solution: Store working aliquots at 4°C for up to one week; for extended storage, conserve at -20°C or -80°C. Avoid repeated freezing and thawing cycles .

These methodological approaches can help overcome the inherent challenges of working with this complex mitochondrial protein and enable more productive research outcomes.

How can researchers effectively validate the functional integrity of recombinant MT-ND4L?

Validating the functional integrity of recombinant MT-ND4L requires a multi-faceted approach that addresses both structural and functional aspects:

• Structural Validation Techniques:

  • Circular Dichroism (CD) Spectroscopy: To confirm proper secondary structure elements expected in membrane proteins.

  • Limited Proteolysis: To assess whether the protein is properly folded, as well-folded proteins typically show resistance to proteolytic digestion at specific sites.

  • Thermal Shift Assays: To evaluate protein stability under various conditions.

• Functional Validation Approaches:

  • Reconstitution Studies: Incorporating recombinant MT-ND4L into liposomes or nanodiscs along with other essential Complex I components to assess proton translocation activity.

  • Proton Translocation Assays: Using pH-sensitive dyes or electrodes to measure proton movement across membranes containing reconstituted MT-ND4L.

  • Comparative Analysis with Native Complex: Comparing the properties of recombinant MT-ND4L with those observed in the native complex isolated from mitochondria.

• Molecular Dynamics Validation:

  • Simulation Comparisons: Comparing the behavior of recombinant protein in silico with established models of MT-ND4L function.

  • Water Channel Analysis: Assessing the formation and maintenance of water channels essential for proton translocation, as mutations have been shown to disrupt these pathways .

• Interaction Studies:

  • Co-Immunoprecipitation: To verify interactions with other Complex I subunits.

  • Crosslinking Studies: To identify specific amino acid residues involved in protein-protein interactions within Complex I.

• Metabolomic Correlation:

  • Functional Impact on Metabolite Profiles: Assessing whether recombinant MT-ND4L variants reproduce the metabolite ratio changes observed in genome-wide association studies, particularly those involving glycerophospholipids .

By employing these complementary validation approaches, researchers can comprehensively assess both the structural integrity and functional capacity of recombinant MT-ND4L, ensuring that experimental findings accurately reflect the protein's native properties and functions.