Recombinant Artemia salina NADH-ubiquinone oxidoreductase chain 4L (ND4L)

What is Artemia salina ND4L and what is its role in mitochondrial function?

Artemia salina NADH-ubiquinone oxidoreductase chain 4L (ND4L) is one of the 13 protein-coding genes in the mitochondrial genome of A. salina. It forms part of Complex I of the mitochondrial electron transport chain, playing a crucial role in cellular respiration and energy production. The gene is encoded in the mitochondrial genome, which in A. salina is 15,762 bp in length with an A+T content of 64.53% . As part of Complex I, ND4L contributes to the transfer of electrons from NADH to ubiquinone, coupled with proton translocation across the inner mitochondrial membrane, which drives ATP synthesis.

How does the ND4L gene in Artemia salina compare to other Artemia species?

The complete mitochondrial genome sequencing of Artemia salina has revealed phylogenetic relationships between different Artemia species. Phylogenetic analysis using the maximum-likelihood method shows that A. salina is positioned at the base of the Artemia phylogenetic tree and has a closer relationship with A. persimilis compared to other Artemia species including A. franciscana, A. urmiana, A. tibetiana, and A. sinica .

The ND4L gene in A. salina, like several other mitochondrial genes, can be analyzed for sequence conservation and evolutionary divergence among Artemia species. Comparative genomic analyses of the ND4L gene sequence can provide insights into the evolutionary history and adaptations of the genus Artemia to extreme environments.

What are the start and stop codons for the ND4L gene in Artemia salina mitochondrial DNA?

While the specific codons for ND4L aren't explicitly stated in the available research, the pattern of codon usage in A. salina mitochondrial genes shows variable start and stop codons. Based on the broader pattern observed in the A. salina mitochondrial genome, several PCGs (protein-coding genes) including cox1, atp6, cox3, cytb, and nd1 use the common ATG start codon . Stop codons include TAA (used by cox3, nd2, atp8, atp6, nd3, and cytb) and TAG (used by nd1, nd4l, and nd6) .

Specifically for ND4L, the research indicates it uses TAG as its stop codon in A. salina . Some mitochondrial genes in A. salina (not specifically stated for ND4L) end with the incomplete stop codon T, which is completed by post-transcriptional polyadenylation.

What are the most effective protocols for isolating and expressing recombinant Artemia salina ND4L?

For isolating and expressing recombinant A. salina ND4L, researchers should consider the following methodological approach:

• DNA Extraction Protocol:

  • Collect A. salina cysts from verified sources (such as those from Sebkha Eladhibet Saltworks, Tunisia)

  • Extract genomic DNA using commercial kits such as TIANGEN®TIANamp Genomic DNA Kit

  • Verify DNA quality through spectrophotometry and gel electrophoresis

• PCR Amplification of ND4L Gene:

  • Design specific primers based on the complete mitochondrial genome sequence (GenBank accession number: MZ199177)

  • Optimize PCR conditions for high specificity and yield

  • Confirm amplicon size and quality through gel electrophoresis

• Cloning and Expression:

  • Clone the ND4L gene into an appropriate expression vector

  • Transform into a suitable expression system (bacterial, yeast, or insect cell systems)

  • Induce protein expression under optimized conditions

  • Purify the recombinant protein using affinity chromatography

Each step requires careful optimization based on the specific research objectives and available resources.

How can I verify the authenticity and functionality of recombinant Artemia salina ND4L?

Verification of recombinant ND4L should follow a multi-step approach:

• Sequence Verification:

  • Perform DNA sequencing of the cloned ND4L gene

  • Compare with the reference sequence (GenBank accession number: MZ199177)

  • Analyze for potential mutations or alterations

• Protein Expression Verification:

  • Western blot analysis using specific antibodies

  • Mass spectrometry for protein identification

  • SDS-PAGE to confirm molecular weight

• Functional Assays:

  • NADH dehydrogenase activity assays

  • Electron transfer measurements

  • Analysis of proton translocation efficiency

• Structural Analysis:

  • Circular dichroism for secondary structure analysis

  • Fluorescence spectroscopy for tertiary structure assessment

  • If possible, structural determination by X-ray crystallography or cryo-EM

These steps ensure both the identity and functional integrity of the recombinant protein.

How can Artemia salina ND4L be utilized in phylogenetic and evolutionary studies?

The ND4L gene from A. salina provides valuable data for phylogenetic and evolutionary studies:

• Phylogenetic Analysis:

  • Comparative sequence analysis with other Artemia species shows that A. salina has a closer relationship with A. persimilis compared to other Artemia species

  • Maximum-likelihood phylogenetic methods with the Kimura 2-parameter model can be employed using software like MEGA 7.0

  • The gene can serve as a molecular marker for resolving relationships among crustacean species

• Evolutionary Rate Studies:

  • Analysis of synonymous and non-synonymous substitution rates in ND4L

  • Comparison of evolutionary rates between different mitochondrial genes

  • Investigation of selection pressures on the ND4L gene in extreme environment adaptation

• Biogeographical Studies:

  • Using ND4L sequence variation to track the dispersal and distribution patterns of Artemia populations

  • Correlation with the "island biogeography" dispersal model mentioned in the research

  • Analysis of genetic divergence in relation to geographical isolation

The complete mitochondrial genome sequence of A. salina, including the ND4L gene, provides essential resources for population genetics research and germplasm conservation .

What is the relationship between Artemia salina ND4L mutations and adaptation to extreme environments?

Artemia species are known extremophiles that inhabit hypersaline environments such as salt lakes and solar saltworks . The relationship between ND4L mutations and environmental adaptation can be studied through:

• Comparative Genomics Approach:

  • Analyze ND4L sequence variations across Artemia populations from different salinity gradients

  • Compare ND4L sequences from populations living in varying temperature conditions

  • Identify potential adaptive mutations through selective pressure analysis

• Structure-Function Correlation:

  • Model the impact of identified mutations on protein structure

  • Analyze how mutations might affect electron transport efficiency

  • Measure how structural changes influence proton pumping under different pH and salinity conditions

• Experimental Validation:

  • Generate recombinant ND4L variants with identified mutations

  • Test functional parameters under varying salt concentrations and pH levels

  • Measure respiratory chain efficiency under conditions mimicking extreme environments

This research direction can provide insights into the molecular mechanisms underlying adaptation to extreme environments at the mitochondrial level.

What are common challenges in expressing functional recombinant Artemia salina ND4L and how can they be overcome?

Researchers often encounter several challenges when working with mitochondrial membrane proteins like ND4L:

• Protein Insolubility:

  • Challenge: Hydrophobic nature of ND4L often leads to inclusion body formation

  • Solution: Use solubility tags (MBP, SUMO), optimize expression conditions (lower temperature, reduced inducer concentration), or employ membrane-mimetic systems (nanodiscs, liposomes)

• Incorrect Folding:

  • Challenge: Achieving native conformation in heterologous expression systems

  • Solution: Co-expression with chaperones, step-wise refolding protocols, or use of specialized expression hosts

• Low Expression Yield:

  • Challenge: Mitochondrial genes often have biased codon usage

  • Solution: Codon optimization for the expression host, use of strong inducible promoters, or selection of appropriate expression strains with rare tRNAs

• Functional Validation:

  • Challenge: Assessing functionality outside the native mitochondrial complex

  • Solution: Reconstitution with other complex I components, development of specialized activity assays, or construction of chimeric proteins

Each challenge requires a systematic approach to optimization based on specific research objectives.

How do post-translational modifications affect recombinant Artemia salina ND4L function and stability?

Post-translational modifications (PTMs) significantly impact ND4L function and stability:

• Types of PTMs Potentially Present:

  • Phosphorylation sites affecting protein-protein interactions

  • Acetylation potentially regulating protein stability

  • Ubiquitination influencing protein turnover

  • Oxidative modifications affecting electron transport function

• Impact on Function:

  • Altered electron transfer efficiency

  • Modified assembly with other Complex I subunits

  • Changed interaction with ubiquinone

  • Variations in proton pumping efficiency

• Detection Methods:

  • Mass spectrometry for comprehensive PTM mapping

  • Western blotting with modification-specific antibodies

  • Phosphoproteomics for phosphorylation site identification

  • Site-directed mutagenesis of predicted PTM sites

• Stabilization Strategies:

  • Optimized buffer conditions to preserve PTMs

  • Strategic mutation of unstable regions

  • Addition of specific cofactors

  • Storage with appropriate protease and phosphatase inhibitors

Understanding these modifications is crucial for producing functionally relevant recombinant protein for research applications.

How does Artemia salina ND4L function compare across different developmental stages and ecological conditions?

The function of ND4L may vary across developmental stages and ecological conditions:

• Developmental Variation:

  • Cyst stage: Potential specialized role during metabolic dormancy

  • Nauplii stage: Support for increased energy demands during early development

  • Adult stage: Adaptation to varying environmental stressors

• Ecological Adaptation:

  • Expression and activity differences in varying salinity conditions

  • Functional modifications in response to temperature fluctuations

  • Structural adaptations to pH variations in hypersaline environments

• Methodology for Comparative Analysis:

  • RT-qPCR for expression level comparison across stages

  • Blue native PAGE for complex assembly analysis

  • Respirometry for functional assessment under varying conditions

  • Proteomics for interactome analysis across developmental stages

This comparative approach provides insights into how ND4L function is regulated to meet varying energetic demands throughout the organism's life cycle and across ecological gradients.

What role might Artemia salina ND4L play in viral susceptibility and pathogen interactions?

Research indicates potential connections between mitochondrial function and viral interactions in Artemia:

• Viral Interaction Mechanisms:

  • Artemia salina can bioaccumulate nervous necrosis virus (NNV) and transmit it to fish larvae

  • Mitochondrial proteins, potentially including ND4L, may be involved in virus-host interactions

  • Possible role in cellular antiviral responses or as targets for viral manipulation

• Experimental Approaches:

  • Co-immunoprecipitation to detect direct interactions between viral proteins and ND4L

  • Gene expression analysis of ND4L during viral infection

  • Functional assays of mitochondrial respiration in infected versus uninfected Artemia

  • Localization studies to track ND4L distribution during infection progression

• Ecological Significance:

  • Potential impact on Artemia's role as disease vectors in aquaculture settings

  • Implications for viral resistance breeding programs

  • Role in horizontal disease transmission dynamics in marine ecosystems

Understanding these interactions has significant implications for aquaculture disease management, as Artemia is widely used as live feed for marine fish larvae .

What emerging technologies might enhance our understanding of Artemia salina ND4L structure and function?

Several cutting-edge technologies hold promise for advancing ND4L research:

• Structural Biology Advances:

  • Cryo-electron microscopy for high-resolution structure determination

  • AlphaFold2 and other AI-based structural prediction tools

  • Single-particle analysis for conformational dynamics studies

  • Hydrogen-deuterium exchange mass spectrometry for protein dynamics

• Functional Genomics Approaches:

  • CRISPR-Cas9 gene editing for in vivo functional studies

  • RNA-Seq for transcriptional network analysis

  • Ribosome profiling for translational regulation studies

  • Single-cell approaches for cell-specific expression analysis

• Biophysical Techniques:

  • Advanced EPR spectroscopy for electron transfer studies

  • Single-molecule FRET for conformational changes

  • Super-resolution microscopy for mitochondrial dynamics

  • Nanoscale respirometry for functional assessment

• Computational Approaches:

  • Molecular dynamics simulations of ND4L in membrane environments

  • Systems biology modeling of respiratory chain function

  • Machine learning for prediction of functional impacts of mutations

  • Multi-omics data integration for comprehensive functional understanding

These technologies will provide unprecedented insights into the structure-function relationships of this important mitochondrial protein.

How might understanding Artemia salina ND4L contribute to biotechnology applications?

Knowledge of A. salina ND4L has several potential biotechnology applications:

• Bioenergy Applications:

  • Engineering more efficient electron transport chains

  • Development of biocatalysts for renewable energy applications

  • Design of biomimetic electron transfer systems

  • Creation of sensors for monitoring environmental toxicants

• Biomedical Applications:

  • Model system for studying mitochondrial disorders

  • Platform for screening therapeutics targeting respiratory chain dysfunction

  • Development of mitochondria-targeted drug delivery systems

  • Understanding mechanisms of hypoxia tolerance with implications for ischemia treatment

• Ecological Applications:

  • Biomarkers for environmental stress monitoring

  • Tools for assessing ecosystem health

  • Indicators for climate change impacts on aquatic ecosystems

  • Development of improved Artemia strains for aquaculture with optimized energy metabolism

• Synthetic Biology Approaches:

  • Creation of minimal synthetic respiratory complexes

  • Development of novel electron transport modules

  • Engineering of stress-resistant mitochondrial systems

  • Design of controllable energy-generating biological systems

The unique adaptations of Artemia to extreme environments make its mitochondrial components, including ND4L, valuable templates for biotechnological innovation.