Recombinant Presbytis melalophos 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 of the mitochondrial genome that encodes the NADH-ubiquinone oxidoreductase chain 4L protein, a critical subunit of NADH dehydrogenase (ubiquinone), also known as Complex I of the electron transport chain . This protein is embedded within the mitochondrial inner membrane and participates in oxidative phosphorylation, the process that generates ATP—the cell's primary energy source .

Complex I functions as the initial enzyme in the electron transport process, facilitating the transfer of electrons from NADH to coenzyme Q10 (ubiquinone) . This electron transfer creates an electrochemical gradient across the inner mitochondrial membrane, which drives ATP synthesis. The MT-ND4L protein specifically contributes to the core hydrophobic domain of Complex I, forming part of the transmembrane region essential for proper complex assembly and function .

Methodologically, researchers investigating MT-ND4L function typically employ mitochondrial isolation protocols followed by respiratory chain complex activity assays to measure electron transfer rates and membrane potential generation in the presence of specific substrates and inhibitors.

What are the structural characteristics of MT-ND4L protein from Presbytis melalophos?

The MT-ND4L protein from Presbytis melalophos is a small protein comprising 98 amino acids with a molecular weight of approximately 11 kDa . The complete amino acid sequence is: MPIIYMNIMLAFTISLLGMLTYRSHLMSSLLCLEGMMLSLFIMSTLMALNMHFPLANIVPIALLVFAACEAAVGLSLLISISNTYGLDHIHNLSLLQC .

This protein is highly hydrophobic, reflecting its function in the transmembrane domain of Complex I . The recombinant form used in research settings typically includes a tag determined during the production process to facilitate purification and detection . The protein's hydrophobic nature necessitates specialized handling protocols, including the use of detergents or lipid environments to maintain proper folding and activity.

For structural studies, researchers commonly employ techniques such as circular dichroism spectroscopy to analyze secondary structure elements, or more advanced methods like cryo-electron microscopy when studying the protein within the larger Complex I assembly.

How does MT-ND4L sequence conservation compare across primate species?

MT-ND4L demonstrates variable conservation across primate species, with specific regions showing higher evolutionary constraint due to their functional importance. Comparative genomic analyses of MT-ND4L in different primate species reveal insights into evolutionary relationships and functional constraints on this mitochondrial protein.

A study analyzing mitochondrial DNA from various Presbytis species, including different subspecies of P. melalophos (femoralis, siamensis, robinsoni, and chrysomelas), demonstrated sequence variations that contribute to phylogenetic understanding . The average sequence divergence within P. melalophos subspecies ranges from 0.00258 (in P. m. chrysomelas) to 0.04139 (in P. m. robinsoni), indicating varying degrees of genetic differentiation .

When conducting comparative sequence analyses, researchers should employ multiple sequence alignment tools followed by calculation of conservation scores at each amino acid position. Evolutionary rate analysis using maximum likelihood methods can identify sites under positive or purifying selection, providing insights into functionally critical regions of the protein.

What experimental approaches are recommended for studying MT-ND4L mutations and their impact on Complex I function?

To investigate MT-ND4L mutations and their functional consequences, researchers should implement a multi-faceted experimental approach:

• Site-directed mutagenesis: Generate recombinant MT-ND4L proteins with specific mutations of interest, such as the T10663C (Val65Ala) mutation associated with Leber hereditary optic neuropathy .

• In vitro reconstitution assays: Incorporate wild-type or mutant MT-ND4L into liposomes with other Complex I subunits to assess assembly efficiency and stability.

• Activity measurements: Employ spectrophotometric assays to measure NADH:ubiquinone oxidoreductase activity, comparing wild-type and mutant proteins under standardized conditions.

• Oxygen consumption analysis: Use high-resolution respirometry to evaluate the impact of mutations on electron transport chain function in intact mitochondria or mitochondrial fractions.

• Structural analysis: Apply cryo-electron microscopy or computational modeling to determine how specific mutations alter protein conformation and interactions within Complex I.

For mutations such as Val65Ala in MT-ND4L, researchers should particularly focus on how the mutation affects:

• Protein stability and half-life

• Complex I assembly efficiency

• Electron transfer rates

• Reactive oxygen species production

• Mitochondrial membrane potential

These comprehensive approaches provide mechanistic insights into how specific MT-ND4L mutations contribute to mitochondrial dysfunction and associated pathologies.

How can recombinant Presbytis melalophos MT-ND4L be effectively utilized in comparative evolutionary studies?

Recombinant Presbytis melalophos MT-ND4L serves as a valuable tool for evolutionary studies through several methodological approaches:

• Molecular phylogenetic analysis: Compare MT-ND4L sequences across primate lineages to reconstruct evolutionary relationships. This approach has successfully differentiated Presbytis subspecies and resolved taxonomic controversies .

• Functional conservation studies: Express recombinant MT-ND4L from different primate species in model systems lacking endogenous protein to assess functional complementation, revealing conservation of biochemical properties.

• Selection pressure analysis: Calculate non-synonymous to synonymous substitution ratios (dN/dS) across different lineages to identify regions under positive or purifying selection.

• Protein interaction comparative studies: Use recombinant proteins to evaluate species-specific differences in interaction patterns with other Complex I subunits.

When applying these approaches, researchers should be aware of methodological considerations including appropriate outgroup selection, accounting for transition/transversion bias (as demonstrated by the Ti/Tv ratio of 9.3 for Cyt b in Presbytis studies) , and integrating nuclear gene data for comprehensive phylogenetic analysis.

What techniques are most effective for studying protein-protein interactions involving MT-ND4L in Complex I assembly?

Investigating protein-protein interactions involving the highly hydrophobic MT-ND4L requires specialized techniques optimized for membrane proteins:

• Chemical cross-linking coupled with mass spectrometry: This approach identifies interaction sites through covalent linkage of neighboring proteins followed by proteomic analysis, particularly valuable for transient interactions within Complex I.

• Co-immunoprecipitation with tagged recombinant proteins: Using epitope-tagged MT-ND4L (as available in recombinant forms) allows for selective pulldown of interaction partners, though the hydrophobic nature of MT-ND4L necessitates careful detergent selection.

• Blue native polyacrylamide gel electrophoresis (BN-PAGE): This technique separates intact protein complexes under non-denaturing conditions, allowing visualization of Complex I assembly intermediates containing MT-ND4L.

• Förster resonance energy transfer (FRET): By tagging MT-ND4L and potential interaction partners with appropriate fluorophores, researchers can detect direct interactions in reconstituted systems or intact mitochondria.

• Surface plasmon resonance (SPR): This biophysical technique measures binding kinetics between immobilized MT-ND4L and other complex I components, providing quantitative interaction parameters.

When implementing these techniques, researchers must carefully consider the hydrophobic nature of MT-ND4L, selecting appropriate detergents or nanodiscs to maintain protein stability while preserving native interactions.

What are the optimal conditions for storage and handling of recombinant MT-ND4L to maintain activity?

Recombinant Presbytis melalophos MT-ND4L requires specific storage and handling conditions to preserve structural integrity and functional activity:

• Storage temperature: Store at -20°C for routine use; for extended storage, maintain at -80°C to prevent degradation .

• Buffer composition: Utilize a Tris-based buffer with 50% glycerol, optimized specifically for this protein to prevent aggregation and maintain solubility .

• Freeze-thaw cycles: Minimize repeated freezing and thawing, which can cause protein denaturation. Working aliquots should be stored at 4°C for up to one week .

• Handling precautions: Due to its hydrophobic nature, include appropriate detergents or lipids in working solutions to prevent aggregation and maintain native conformation.

• Stability assessment: Prior to experimental use, verify protein integrity through techniques such as SDS-PAGE, Western blotting, or activity assays to ensure the protein remains properly folded and functional.

Researchers should perform preliminary stability studies to determine the optimal conditions for their specific experimental applications, as different assay systems may require modified handling protocols.

How can researchers verify the functional activity of recombinant MT-ND4L in experimental systems?

Verifying the functional activity of recombinant MT-ND4L requires multiple complementary approaches:

• NADH:ubiquinone oxidoreductase activity assay: Measure the rate of NADH oxidation spectrophotometrically at 340 nm in the presence of ubiquinone analogs, with successful incorporation of active MT-ND4L resulting in higher electron transfer rates.

• Membrane potential measurements: Use potential-sensitive fluorescent dyes like JC-1 or TMRM to assess whether MT-ND4L incorporation enhances proton pumping activity when reconstituted into liposomes with other Complex I components.

• Supercomplex assembly analysis: Employ blue native gel electrophoresis to determine if recombinant MT-ND4L facilitates the formation of higher-order respiratory chain supercomplexes.

• Complementation studies: Introduce recombinant MT-ND4L into cellular systems with MT-ND4L deficiencies to assess functional rescue of respiratory chain activity and ATP production.

• Structural incorporation verification: Use proteoliposome flotation assays or limited proteolysis to confirm proper integration of recombinant MT-ND4L into membranes or Complex I subcomplexes.

A systematic evaluation using these techniques provides comprehensive evidence of functional activity, ensuring experimental reliability for downstream applications.

What methodological approaches are recommended for investigating MT-ND4L involvement in mitochondrial disease models?

Investigating MT-ND4L's role in mitochondrial diseases requires integrated methodological approaches:

• Patient-derived cell models: Establish fibroblast or induced pluripotent stem cell (iPSC) lines from patients with MT-ND4L mutations, such as the T10663C mutation associated with Leber hereditary optic neuropathy (LHON) .

• CRISPR-based mitochondrial DNA editing: Though technically challenging, emerging techniques for mitochondrial DNA editing allow introduction of specific MT-ND4L mutations to create isogenic cell lines for comparative studies.

• Cybrid (cytoplasmic hybrid) technology: Transfer patient mitochondria containing MT-ND4L mutations into cells depleted of mitochondrial DNA to isolate the effect of mitochondrial mutations from nuclear genetic background.

• Tissue-specific phenotyping: Since MT-ND4L mutations may affect tissues differentially (as seen in LHON's specific impact on retinal ganglion cells) , develop differentiation protocols to generate affected cell types from patient-derived iPSCs.

• Therapeutic strategy testing: Use established disease models to evaluate potential interventions, including:

  • Gene therapy approaches

  • Small molecules enhancing Complex I activity

  • Bypass strategies to circumvent Complex I deficiency

These approaches should incorporate comprehensive bioenergetic profiling measuring oxygen consumption, ATP production, and reactive oxygen species generation to characterize the pathogenic mechanisms of MT-ND4L mutations.

How does MT-ND4L sequence variation correlate with phylogenetic relationships among primate species?

MT-ND4L sequence variations provide valuable insights into primate phylogenetic relationships, though the analysis requires careful methodological consideration:

Phylogenetic analysis of MT-ND4L sequences, along with other mitochondrial genes like cytochrome b and 12S rRNA, has helped resolve taxonomic controversies among Presbytis species and subspecies . Studies of P. melalophos subspecies (femoralis, siamensis, robinsoni, and chrysomelas) revealed varying degrees of genetic divergence, with sequence divergence values ranging from 0.00258 to 0.04139 .

When conducting phylogenetic analyses using MT-ND4L:

• Account for nucleotide substitution biases: The transition/transversion ratio in mitochondrial genes can be quite high (9.3 for Cyt b in Presbytis studies) , requiring appropriate substitution models.

• Employ multiple phylogenetic reconstruction methods: Combine Neighbor-Joining (NJ), Maximum Parsimony (MP), and Maximum Likelihood (ML) approaches to obtain robust phylogenetic trees.

• Assess node support rigorously: Implement bootstrap resampling (minimum 1000 replicates) to evaluate the stability of tree topologies .

• Integrate multiple genetic markers: Combine MT-ND4L data with other mitochondrial and nuclear markers for comprehensive phylogenetic analysis.

• Consider functional constraints: Analyze whether conservation patterns correlate with functional domains of the MT-ND4L protein.

This methodological framework allows researchers to accurately interpret MT-ND4L sequence variations in the context of primate evolution and speciation.

What analytical tools and statistical approaches are recommended for detecting selection pressure on MT-ND4L across species?

Detecting selection pressure on MT-ND4L requires specialized analytical approaches that account for the unique properties of mitochondrial genes:

• Codon-based selection analysis: Implement PAML (Phylogenetic Analysis by Maximum Likelihood) or HyPhy to calculate dN/dS ratios at individual codons, identifying sites under positive or purifying selection.

• Sliding window analysis: Apply this approach to detect localized regions of the gene under different selection pressures, particularly relevant for the functionally distinct domains of MT-ND4L.

• McDonald-Kreitman test: Compare polymorphism within species to divergence between species to detect adaptive evolution signatures.

• Functional divergence analysis: Use software like DIVERGE to identify amino acid positions showing functional divergence between different evolutionary lineages.

• Structural mapping of selection: Map selected sites onto 3D structural models of MT-ND4L to determine whether selection correlates with specific structural or functional domains.

When implementing these analyses for MT-ND4L, researchers should account for:

• The overlapping gene structure with MT-ND4 (7-nucleotide overlap) , which can constrain evolutionary changes

• Mitochondrial genetic code variations

• Maternal inheritance pattern of mitochondrial DNA

• Potential effects of demographic history on selection inference

These methodological considerations ensure robust detection of evolutionary pressures shaping MT-ND4L diversity across primate species.

How can researchers integrate recombinant MT-ND4L into functional studies of mitochondrial respiratory complexes?

Integrating recombinant MT-ND4L into functional studies requires specialized approaches that address the challenges of working with hydrophobic membrane proteins:

• Proteoliposome reconstitution: Incorporate purified recombinant MT-ND4L into artificial lipid bilayers along with other Complex I subunits to create minimal functional systems. This approach requires:

  • Optimized lipid composition mimicking the mitochondrial inner membrane

  • Careful protein-to-lipid ratio determination

  • Verification of correct orientation in the membrane

• Nanodiscs technology: Assemble MT-ND4L with other Complex I components into nanodiscs (disc-shaped phospholipid bilayers stabilized by scaffold proteins), providing a native-like environment while maintaining solubility and accessibility for functional studies.

• Cell-free expression systems: Utilize cell-free protein synthesis approaches optimized for membrane proteins to express MT-ND4L directly into artificial membranes or detergent micelles.

• Complementation studies: Introduce recombinant MT-ND4L into cell lines with MT-ND4L deficiencies, then assess restoration of:

  • Complex I assembly (via blue native PAGE)

  • NADH:ubiquinone oxidoreductase activity

  • Mitochondrial respiration (via high-resolution respirometry)

  • ATP production capacity

  • Reactive oxygen species generation

These approaches enable researchers to dissect the specific contributions of MT-ND4L to Complex I function, assembly, and stability under controlled experimental conditions.

What are the critical considerations when designing experiments to study the unusual gene overlap between MT-ND4L and MT-ND4?

The 7-nucleotide overlap between MT-ND4L and MT-ND4 genes represents an intriguing feature requiring specialized experimental approaches:

• Transcriptional analysis considerations: When studying MT-ND4L/MT-ND4 expression, design RNA-seq or qRT-PCR experiments that can distinguish the overlapping region where MT-ND4L's last three codons (5'-CAA TGC TAA-3' coding for Gln, Cys and Stop) overlap with MT-ND4's first three codons (5'-ATG CTA AAA-3' coding for Met-Leu-Lys) .

• Mutagenesis strategy design: When introducing mutations for functional studies, carefully analyze potential effects on both reading frames:

  • Mutations in the +1 reading frame (MT-ND4L) may affect the +3 reading frame (MT-ND4)

  • Silent mutations in one gene may create non-synonymous changes in the overlapping gene

• Evolutionary constraint analysis: Study selection pressures on this overlapping region, as mutations must be compatible with both proteins, creating unique evolutionary constraints.

• Translation coordination experiments: Design ribosome profiling experiments to investigate whether the overlapping region influences translation efficiency or coordination between the two genes.

• Structural interaction studies: Investigate whether the proteins encoded by these overlapping genes physically interact in the assembled Complex I, potentially explaining the evolutionary conservation of this arrangement.

These methodological considerations enable researchers to address fundamental questions about the functional significance of this unusual gene organization in mitochondrial genome evolution.