Recombinant Phalanger vestitus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

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

MT-ND4L (mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4L) is a core subunit of mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). It belongs to the minimal assembly required for catalysis in the electron transport chain. Its primary function involves the transfer of electrons from NADH to the respiratory chain, with ubiquinone serving as the immediate electron acceptor . The protein is encoded by the mitochondrial genome and plays a crucial role in the oxidative phosphorylation process that generates cellular ATP.

MT-ND4L dysfunction can lead to energy deficiency in cells, potentially resulting in various metabolic disorders. Research has shown that changes in MT-ND4L gene expression have long-term consequences on energy metabolism and may be a major predisposition factor for the development of metabolic syndrome .

How does recombinant MT-ND4L differ from the native protein?

Recombinant MT-ND4L is artificially produced in expression systems (commonly E. coli) rather than isolated from natural sources. The major differences include:

• Presence of affinity tags: Recombinant proteins often contain tags (such as His-tags) to facilitate purification .

• Expression environment: Recombinant proteins are produced in heterologous systems rather than in mitochondria, which may affect post-translational modifications.

• Storage conditions: Recombinant proteins are typically provided in specialized buffers (e.g., Tris-based buffer with 50% glycerol) optimized for stability .

• Potential structural variations: The recombinant protein may have slight conformational differences from the native protein due to the expression environment and the absence of natural assembly partners.

These differences must be considered when designing experiments utilizing recombinant MT-ND4L for functional studies.

How can recombinant Phalanger vestitus MT-ND4L be used to study mitochondrial disease mechanisms?

Recombinant Phalanger vestitus MT-ND4L can serve as a valuable tool for investigating mitochondrial disease mechanisms through several experimental approaches:

• In vitro reconstitution studies: Researchers can use the recombinant protein to reconstitute Complex I activity in controlled environments, allowing the assessment of how specific mutations affect electron transport .

• Protein-protein interaction analyses: The recombinant protein enables the identification of interaction partners in Complex I assembly and function. Based on similar studies with ovine Complex I, MT-ND4L likely interacts with various subunits within the N-module (NDUFV1, NDUFV2, NDUFV3-10, NDUFS1, NDUFA12) and Q-module (NDUFS3, NDUFS8, NDUFA6, NDUFA9) .

• Structural studies: Purified recombinant protein can be used for structural analyses to determine how mutations affect protein folding and complex assembly.

• Antibody production: The recombinant protein can be used to generate specific antibodies for immunodetection of the native protein in tissue samples from patients with suspected mitochondrial disorders.

• Metabolomic correlation studies: As demonstrated in the research on MT-ND4L variants and metabolite ratios, recombinant proteins can help validate the functional consequences of specific mutations on metabolite profiles .

What are the key considerations for experimental design when studying MT-ND4L function?

When designing experiments to study MT-ND4L function, researchers should consider several critical factors:

• Membrane integration: As a highly hydrophobic protein, MT-ND4L requires appropriate membrane environments or detergent systems for functional studies.

• Complex assembly: MT-ND4L functions as part of Complex I, so isolated protein studies must consider the absence of interaction partners and the complex assembly process.

• Redox environment: The protein functions in electron transfer, necessitating controlled redox conditions for accurate functional assessment.

• Post-translational modifications: Evidence suggests MT-ND4L may be regulated by phosphorylation. Researchers should consider how expression systems might affect these modifications .

• Species-specific differences: When using Phalanger vestitus MT-ND4L as a model, researchers must account for potential functional differences compared to human or other species' orthologs.

Table 1: Comparison of experimental approaches for MT-ND4L functional studies

How can researchers optimize the expression and purification of recombinant Phalanger vestitus MT-ND4L?

Optimizing expression and purification of recombinant Phalanger vestitus MT-ND4L presents several challenges due to its hydrophobic nature and mitochondrial origin. Based on the available information for similar proteins, here is a methodological approach:

• Expression system selection:

  • E. coli systems are commonly used (as seen in the search results for related proteins)

  • Consider membrane-protein specialized strains like C41(DE3) or C43(DE3)

  • For complex post-translational modifications, eukaryotic systems may be preferable

• Expression optimization:

  • Lower induction temperatures (16-20°C) to facilitate proper folding

  • Use fusion partners (e.g., MBP, SUMO, or thioredoxin) to enhance solubility

  • Codon optimization for the expression host

• Purification strategy:

  • Affinity purification using N-terminal or C-terminal His-tags

  • Detergent screening (LDAO, DDM, or Triton X-100) for membrane protein extraction

  • Size exclusion chromatography for final polishing

• Stability considerations:

  • Store in Tris-based buffer with glycerol (typically 50%) as indicated in product specifications

  • Avoid repeated freeze-thaw cycles

  • Consider aliquoting for single use

• Quality control:

  • SDS-PAGE analysis (aim for >90% purity)

  • Mass spectrometry confirmation

  • Functional assays to confirm activity

What techniques are most effective for studying the interactions between MT-ND4L and other Complex I subunits?

Investigating protein-protein interactions involving MT-ND4L requires specialized approaches due to its membrane-embedded nature. The most effective techniques include:

• Crosslinking mass spectrometry (XL-MS):

  • Identifies specific residues involved in subunit interactions

  • Can be performed in native membrane environments

  • Provides spatial constraints for structural modeling

• Co-immunoprecipitation with tagged constructs:

  • Allows for pull-down of interaction partners

  • Can be combined with mass spectrometry for unbiased identification

  • Requires careful control for detergent effects on interactions

• Proximity labeling approaches (BioID or APEX2):

  • Labels proteins in close proximity in living cells

  • Effective for transient or weak interactions

  • Can identify novel interaction partners

• Cryo-electron microscopy:

  • Provides structural context for interactions

  • Can visualize the protein in the context of the entire Complex I

  • Requires substantial technical expertise and equipment

• Förster resonance energy transfer (FRET):

  • Measures interactions in live cells or reconstituted systems

  • Can detect conformational changes during function

  • Requires careful fluorophore placement to avoid functional disruption

Current evidence from related studies indicates MT-ND4L likely interacts with various subunits within the N-module and Q-module of Complex I, including NDUFV1, NDUFV2, NDUFS1, NDUFS3, and NDUFS8 .

How do mutations in MT-ND4L contribute to mitochondrial dysfunction and disease?

Mutations in MT-ND4L can significantly impact mitochondrial function through several mechanisms:

The search results specifically note that "Changes in MT-ND4L gene expression have long-term consequences on energy metabolism and have been suggested to be a major predisposition factor for the development of metabolic syndrome" . Additionally, "several variants of human MT-ND4L have been reported to be associated with altered metabolic conditions like BMI and type 2 diabetes" .

What is the significance of the MT-ND4L variant mt10689 G>A and its association with metabolic profiles?

The variant mt10689 G>A in the MT-ND4L gene has been identified as having significant associations with metabolite ratios, particularly those involving phosphatidylcholine diacyl C36:6 (PC aa C36:6) . This finding has several important implications:

• Metabolic pathway connections: This variant shows the largest number of significant associations between metabolite ratios and mitochondrial SNVs, suggesting it plays a critical role in metabolic regulation.

• Phosphatidylcholine metabolism: PC aa C36:6 was involved in 16 different metabolite ratios associated with this variant, highlighting a potential mechanistic link between MT-ND4L function and phospholipid metabolism.

• Fat metabolism correlations: PC aa C36:6 has been associated with different patterns of fat concentration in the body, including visceral fat and liver fat content , connecting this variant to potential metabolic disorder mechanisms.

• Clinical relevance: The metabolite PC aa C36:6 was also involved in three metabolite ratios previously shown to be associated with Fat-Free Mass Index , suggesting potential applications in metabolic disorder diagnostics.

These associations provide insight into how mitochondrial genetic variation in MT-ND4L may influence metabolic health through alterations in phospholipid metabolism and energy homeostasis.

How can researchers effectively utilize MT-ND4L in metabolomic studies?

Integrating MT-ND4L analysis with metabolomics offers powerful insights into mitochondrial function and metabolic regulation. Based on the search results, here is a methodological approach:

• Study design considerations:

  • Combine genotyping for MT-ND4L variants with targeted metabolomics

  • Include metabolite ratios as they may be more informative than absolute concentrations

  • Account for confounding variables such as age and sex in statistical models

• Analytical approach:

  • Follow the "inverted mtGWAS" approach described in the literature, where genetic variants are used as outcome variables and metabolite ratios as predictors

  • The model can be represented as: mtSNV(i,k) = β0(i) + β1(i) × metabolite_ratio(j,k) + β2(i) × age(k) + β3(i) × sex(k) + ε(i,j,k)

  • This approach evaluates how mitochondrial heteroplasmy is influenced by metabolite ratios

• Key metabolites to monitor:

  • Focus on phosphatidylcholines, particularly PC aa C36:6, which has shown strong associations with MT-ND4L variants

  • Consider metabolite ratios that reflect specific metabolic pathways

• Data interpretation framework:

  • Look for patterns of association that may indicate metabolic pathway disruptions

  • Consider the physiological context of identified associations

  • Validate findings with functional studies using recombinant proteins

This approach can reveal how genetic variations in MT-ND4L impact metabolic health and potentially identify novel biomarkers for mitochondrial dysfunction.

What are the best approaches for studying post-translational modifications of MT-ND4L?

Post-translational modifications (PTMs) of MT-ND4L, particularly phosphorylation, may play critical roles in regulating its function. Based on the search results, MT-ND4L contains predicted phosphorylation sites for AMP-activated protein kinase A (PKA) . Studying these modifications requires specialized approaches:

• PTM site identification:

  • Targeted mass spectrometry using multiple reaction monitoring (MRM)

  • Phospho-specific antibodies (if available)

  • In silico prediction followed by site-directed mutagenesis

• Functional impact assessment:

  • Compare wild-type and phospho-mimetic mutants (e.g., Ser to Asp/Glu) in activity assays

  • Analyze effects on protein-protein interactions and Complex I assembly

  • Measure impact on electron transfer efficiency

• Regulatory mechanisms:

  • Investigate kinase/phosphatase dynamics (particularly PKA) in response to cellular energy status

  • Study the impact of energy stress on MT-ND4L phosphorylation state

  • Analyze tissue-specific patterns of MT-ND4L modification

• Technical considerations:

  • Preserve labile modifications during sample preparation

  • Use phosphatase inhibitors when appropriate

  • Consider native versus recombinant protein contexts

Current evidence suggests that PKA-mediated phosphorylation of MT-ND4L may affect:

• Mitochondrial import and MTS removal

• Incorporation into Complex I

• Complex I activity stimulation

These processes are critical for understanding how MT-ND4L function is regulated in response to changing cellular conditions.

What are the emerging research areas for MT-ND4L and what technical advances might facilitate new discoveries?

Research on MT-ND4L is evolving rapidly, with several promising directions for future investigation:

• Single-cell energetics: Emerging technologies for single-cell analysis of mitochondrial function may reveal cell-type specific roles of MT-ND4L variants in metabolic regulation.

• Integration with multi-omics data: Combining MT-ND4L genetic information with proteomics, metabolomics, and transcriptomics can provide a systems-level understanding of its role in health and disease.

• Therapeutic targeting: As understanding of MT-ND4L function improves, it may become a target for interventions in metabolic disorders.

• Evolutionary perspectives: Comparative studies of MT-ND4L across species (including Phalanger vestitus) may reveal evolutionary adaptations in energy metabolism.

• Environmental interactions: Investigating how environmental factors interact with MT-ND4L variants to influence metabolic health represents an important frontier.

Technical advances that will facilitate these investigations include improvements in cryo-EM resolution for membrane protein complexes, more sensitive metabolomic profiling techniques, and CRISPR-based approaches for introducing mitochondrial DNA modifications.