Recombinant Lipotes vexillifer NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the function of MT-ND4L in cellular energy metabolism?

MT-ND4L (NADH dehydrogenase 4L) is a crucial component of complex I (NADH:ubiquinone oxidoreductase) in the mitochondrial respiratory chain. This protein participates in the first step of electron transport during oxidative phosphorylation, transferring electrons from NADH to ubiquinone across the inner mitochondrial membrane . This electron transfer creates an electrochemical gradient that drives ATP synthesis, making MT-ND4L essential for cellular energy production. The protein is embedded in the hydrophobic domain of complex I within the inner mitochondrial membrane, where it contributes to the complex's proton-pumping activity .

Experimental approach: Researchers can assess MT-ND4L function through complex I activity assays measuring NADH oxidation rates spectrophotometrically. Oxygen consumption measurements using respirometry can also evaluate the protein's contribution to mitochondrial energy production in intact cells or isolated mitochondria.

How does one verify the expression and localization of recombinant MT-ND4L?

Verification of recombinant MT-ND4L expression requires multiple complementary approaches:

• Western blotting with antibodies specific to MT-ND4L or fusion tags

• Immunofluorescence microscopy for cellular localization

• Subcellular fractionation followed by Western blot analysis

• Mass spectrometry for protein identification and post-translational modification analysis

For proper mitochondrial localization assessment, researchers can use mitochondrial markers (such as MitoTracker dyes) in conjunction with immunofluorescence of the recombinant protein. Both confocal microscopy and super-resolution techniques provide detailed information on the precise localization within mitochondria . Successful mitochondrial targeting should show co-localization with established mitochondrial markers, particularly in the inner membrane fraction.

What expression systems are optimal for recombinant Lipotes vexillifer MT-ND4L production?

The expression of mitochondrial membrane proteins like MT-ND4L presents significant challenges. Based on related mitochondrial protein expression studies, the following systems show promise:

*Note: E. coli yields are typically higher but often require refolding from inclusion bodies for membrane proteins.

For functional studies, mammalian cell expression using adeno-associated viral vectors has shown success in expressing mitochondrial proteins while maintaining their functionality .

How do mutations in MT-ND4L affect complex I assembly and function?

Mutations in MT-ND4L can significantly impact complex I assembly and function, as evidenced by studies on related NADH dehydrogenase subunits. The T10663C (Val65Ala) mutation in MT-ND4L has been associated with Leber hereditary optic neuropathy , suggesting critical functional importance of this residue.

Methodological approach for investigating mutation effects:

• Site-directed mutagenesis of recombinant MT-ND4L

• Blue Native PAGE to assess complex I assembly

• Respirometry to measure functional impact on electron transport

• Reactive oxygen species (ROS) detection to evaluate secondary effects

• Molecular dynamics simulations to predict structural changes

Researchers should employ transmitochondrial cybrid cell lines, where patient-derived mitochondria with specific mutations are introduced into cells depleted of mitochondrial DNA. This allows for controlled study of mutation effects in a consistent nuclear genetic background .

What techniques effectively characterize MT-ND4L conformational dynamics?

Understanding MT-ND4L dynamics requires advanced biophysical and computational approaches:

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS) provides insights into protein flexibility and solvent accessibility

• Advanced AI-driven conformational ensemble generation as described in recent research:

  • Prediction of alternative functional states along collective coordinates

  • Molecular simulations with AI-enhanced sampling

  • Trajectory clustering to identify representative structures

  • Diffusion-based AI models to generate statistically robust conformational ensembles

• Molecular dynamics simulations with specialized force fields for membrane proteins to model behavior in lipid environments

• Cysteine crosslinking experiments to validate computational predictions of conformational changes

Researchers should combine these techniques to develop a comprehensive understanding of MT-ND4L dynamics, particularly focusing on how conformational changes might influence electron transfer efficiency in complex I .

How can binding pockets in MT-ND4L be identified and characterized for therapeutic development?

Identification of binding pockets in MT-ND4L requires a multi-faceted approach combining computational prediction and experimental validation:

• AI-based pocket prediction algorithms incorporating protein dynamics information generated from conformational ensemble analysis

• Ensemble-based pocket detection algorithms that utilize previously established protein dynamics to identify transient pockets that may not be visible in static structures

• Integration of LLM-driven literature search data with structure-aware detection methods to leverage existing binding site information across homologous proteins

• Small molecule probe screening using libraries of fluorescent or covalent probes to experimentally validate predicted pockets

• Characterization through fragment-based screening approaches to determine druggability of identified pockets

This comprehensive approach has successfully identified orthosteric, allosteric, hidden, and cryptic binding pockets on protein surfaces , providing potential targets for therapeutic development against complex I dysfunction associated with neurodegenerative disorders .

What is the role of MT-ND4L in neurodegenerative disorders and potential therapeutic approaches?

Complex I dysfunction, including MT-ND4L abnormalities, has been implicated in several neurodegenerative conditions:

• Parkinson's disease: Complex I inhibition by toxins like rotenone and pyridaben induces Parkinson's-like symptoms, suggesting complex I dysfunction in pathogenesis

• Huntington's disease: Mitochondrial dysfunction involving complex I contributes to neurodegeneration

• Multiple sclerosis: Novel mutations in related complex I components have been identified in MS patients, suggesting mitochondrial dysfunction plays a role in disease progression

Therapeutic approach: One promising strategy involves complementation with alternative NADH dehydrogenases. Research has demonstrated that the single-subunit NADH dehydrogenase from Saccharomyces cerevisiae (Ndi1) can functionally replace complex I in mammalian cells, conferring resistance to complex I inhibitors. When expressed in dopaminergic cell lines using adeno-associated virus vectors, Ndi1:

• Localized to both cell bodies and neurites

• Maintained functional activity

• Allowed cells to undergo normal morphological maturation

• Provided resistance to complex I inhibitors

This approach represents a potential gene therapy strategy for neurodegenerative conditions caused by complex I dysfunction, which could be applied to disorders involving MT-ND4L mutations.

What post-translational modifications affect Lipotes vexillifer MT-ND4L function?

Post-translational modifications (PTMs) of MT-ND4L can significantly influence its function within complex I. Research on mitochondrial-encoded subunits of complex I has identified several critical PTMs:

• N-α-formylation: Mitochondrial-encoded subunits, including those homologous to Lipotes vexillifer MT-ND4L, retain their N-α-formyl methionine residues after translation using the mitochondrial genetic code

• Acetylation: While some nuclear-encoded complex I subunits are N-α-acetylated, this modification appears less conserved across species

• Phosphorylation: Serine, threonine, and tyrosine phosphorylation can regulate complex I activity

Methodological approach for PTM identification:

• Immunoprecipitation of recombinant or native MT-ND4L

• Mass spectrometry analysis using both:

  • Peptide mass fingerprinting

  • Tandem MS for modification mapping

• Molecular mass measurements of intact protein to detect changes from theoretical mass

Researchers investigating Lipotes vexillifer MT-ND4L should use a combination of protein separation methods (SDS-PAGE and HPLC) coupled with multiple mass spectrometry techniques for comprehensive PTM characterization, as this approach has proven necessary for thorough analysis of complex I subunits in other species .

How does MT-ND4L integrate into the complete complex I structure?

MT-ND4L integrates into complex I as one of the core subunits within the membrane domain. Complex I is a large, multi-subunit enzyme with a characteristic L-shaped structure consisting of a hydrophilic arm extending into the mitochondrial matrix and a hydrophobic arm embedded in the inner mitochondrial membrane.

Integration process:

• MT-ND4L is synthesized within the mitochondria from the mitochondrial genome

• The protein inserts into the inner mitochondrial membrane through the mitochondrial insertion machinery

• MT-ND4L associates with other membrane-embedded subunits during the assembly of the membrane arm

• The fully assembled membrane domain then connects with the matrix arm to form the complete complex I

In comprehensive proteomic analyses of complex I from various species, MT-ND4L has been identified as one of the 14 core (conserved) subunits, with seven of these core subunits being mitochondrially encoded . The protein plays a critical role in the proton-pumping function of complex I during electron transfer from NADH to ubiquinone.

What experimental approaches can assess the interaction between recombinant MT-ND4L and other complex I subunits?

Investigating subunit interactions within complex I requires specialized techniques due to the hydrophobic nature of many components:

• Chemical crosslinking coupled with mass spectrometry (XL-MS)

  • Utilizes bifunctional crosslinking reagents to capture protein-protein interactions

  • MS analysis identifies crosslinked peptides, revealing proximity relationships

  • Data can be used to generate spatial constraints for structural modeling

• Blue Native PAGE combined with second-dimension SDS-PAGE

  • Separates intact complexes in the first dimension while preserving native interactions

  • Second dimension separates individual subunits

  • Western blotting identifies specific interaction partners

• Förster Resonance Energy Transfer (FRET)

  • Requires fluorescent labeling of MT-ND4L and potential interaction partners

  • Provides dynamic information about protein interactions in living cells

  • Particularly useful for monitoring assembly intermediates

• Co-immunoprecipitation with tagged recombinant proteins

  • Allows pull-down of interaction partners when antibodies against native proteins are unavailable

  • Can identify weak or transient interactions that may occur during assembly

• Proximity-dependent biotin labeling (BioID or APEX)

  • Fusion of biotin ligase to MT-ND4L labels proximal proteins

  • Labeled proteins are identified by streptavidin pull-down and mass spectrometry

  • Maps the protein neighborhood in living cells

These techniques have been successfully applied to characterize the composition and assembly of complex I from various species, revealing that the enzyme typically comprises 35-41 subunits depending on the organism .

How can recombinant MT-ND4L be utilized for drug discovery targeting mitochondrial dysfunction?

Recombinant MT-ND4L can serve as a valuable tool for drug discovery through several approaches:

• High-throughput screening platforms

  • Incorporation of recombinant MT-ND4L into liposomes or nanodiscs

  • Development of activity assays suitable for compound screening

  • FRET-based interaction assays to identify compounds that modulate protein-protein interactions

• Structure-based drug design

  • Utilization of AI-driven conformational ensemble generation to identify multiple protein states

  • Virtual screening against identified binding pockets

  • Fragment-based drug discovery to develop lead compounds

• Phenotypic rescue screening

  • Expression of mutant MT-ND4L variants in cellular models

  • Screening for compounds that restore mitochondrial function

  • Identification of therapeutic candidates for diseases like Leber hereditary optic neuropathy

• Alternative NADH dehydrogenase complementation

  • Using single-subunit NADH dehydrogenases (like Ndi1) as an alternative approach

  • Screening for compounds that enhance expression or activity of these complementary proteins

  • Development of gene therapy approaches for neurodegenerative disorders

The identification of binding pockets through AI-based prediction methods combined with structure-aware ensemble-based detection algorithms provides a robust foundation for targeted drug discovery efforts .

What are the challenges in expressing and studying Lipotes vexillifer MT-ND4L compared to other mammalian homologs?

The Yangtze River dolphin (Lipotes vexillifer) MT-ND4L presents unique challenges compared to other mammalian homologs:

• Limited reference material

  • Near-extinction status of the species limits availability of fresh tissue samples

  • Reliance on archived specimens or genomic databases

  • Challenges in validating expression constructs against native protein

• Codon optimization considerations

  • Potential unique codon usage patterns in Lipotes vexillifer

  • Need for careful construct design for expression in various host systems

  • Optimization requirements may differ between bacterial and eukaryotic expression systems

• Species-specific post-translational modifications

  • Potential unique PTMs in aquatic mammals adapted to hypoxic conditions

  • Challenges in reproducing these modifications in recombinant systems

  • Need for comprehensive PTM mapping using advanced mass spectrometry

• Functional characterization challenges

  • Difficulty in establishing appropriate reference values for activity

  • Limited knowledge of species-specific interaction partners

  • Challenges in interpreting functional data without species-matched components

Methodological approach: Researchers should employ comparative studies with well-characterized homologs (e.g., human, bovine) while using sensitive analytical techniques capable of detecting subtle structural and functional differences. Homology modeling based on high-resolution structures of other mammalian complex I can provide valuable structural insights prior to experimental characterization.

What is the current state of research on MT-ND4L and future research priorities?

The current state of research on MT-ND4L reveals its critical importance in mitochondrial function and association with several human diseases. Key findings include:

• MT-ND4L functions as an essential component of complex I in the electron transport chain, participating in the first step of oxidative phosphorylation

• Mutations in MT-ND4L are associated with Leber hereditary optic neuropathy, while dysfunction of complex I more broadly is implicated in neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and potentially multiple sclerosis

• Advanced AI-driven approaches have enhanced our ability to study protein conformational dynamics and identify potential binding pockets for therapeutic development

• Expression of alternative NADH dehydrogenases shows promise as a therapeutic strategy for complex I deficiencies

Future research priorities should focus on:

• Developing improved expression systems for recombinant production of functional MT-ND4L for structural and functional studies

• Expanding our understanding of species-specific variations in MT-ND4L structure and function, particularly in endangered species like Lipotes vexillifer

• Applying advanced computational methods to predict the impact of mutations and design targeted therapeutics

• Exploring gene therapy approaches using alternative NADH dehydrogenases or gene editing technologies to address MT-ND4L deficiencies

• Investigating the role of MT-ND4L in adaptive responses to environmental stressors, particularly in aquatic mammals