Recombinant Mouse Leucine--tRNA ligase, cytoplasmic (Lars), partial

What is the basic structure and function of mouse LARS?

Mouse LARS is a large multidomain aminoacyl-tRNA synthetase responsible for catalyzing the attachment of leucine to its cognate tRNA molecules for protein translation. The protein contains several functional domains including a catalytic Rossman fold, leucine-specific domains (LSD), and regions involved in protein-protein interactions. LARS functions both as an essential enzyme for protein synthesis and as a leucine sensor in cellular signaling pathways .

The protein has two primary functions:

• Catalytic activity: Aminoacylation of tRNA^Leu for protein translation

• Signaling function: Acting as a leucine sensor for the mTOR pathway, interacting with both RagD GTPase and Vps34 lipid kinase to promote mTORC1 signaling

These dual roles make LARS a particularly interesting subject for research at the intersection of translation and cellular signaling mechanisms.

What are the known splice variants of mouse LARS and how do they differ functionally?

Long-read sequencing has identified multiple LARS splice variants (LSVs) with distinct functional properties. The most extensively characterized is LSV3, which has a 71 amino acid deletion in the catalytic domain resulting from exon 20 skipping. This deletion has significant functional consequences:

The LSV3 variant demonstrates a remarkable evolutionary separation between the ancient catalytic function and the newer signaling role. Despite lacking aminoacylation activity, LSV3 maintains its interactions with mTOR pathway components, including RagD GTPase, although the deletion includes part of the previously mapped Vps34-binding domain .

How does LARS participate in the mTOR signaling pathway?

LARS serves as an intracellular leucine sensor for the mTOR pathway through two distinct mechanisms:

• RagD-dependent pathway: LARS binds leucine using the same site as in catalysis, which triggers a conformational change that enhances its interaction with RagD GTPase. This facilitates GTP hydrolysis by RagD, converting it to GDP-RagD, which then promotes mTORC1 kinase activity .

• Vps34-dependent pathway: LARS binds to and activates Vps34 lipid kinase, which provides an orthogonal signaling route to activate mTORC1 .

Interestingly, even the catalytically inactive LSV3 splice variant maintains these signaling capabilities, suggesting that the amino acid deletion in exon 20 does not significantly disrupt the protein-protein interactions required for mTOR signaling .

What are the optimal conditions for recombinant expression of mouse LARS?

For efficient expression of recombinant mouse LARS, researchers should consider the following methodological approach:

• Expression system: E. coli is commonly used for expressing recombinant LARS proteins. Based on similar approaches for related proteins, BL21(DE3) or Rosetta(DE3) strains are recommended for higher expression yields .

• Vector design: Incorporate a histidine tag (6xHis) at either the N- or C-terminus to facilitate purification, similar to the approach used for mouse LRPAP protein .

• Induction parameters:

  • Temperature: 18°C after induction

  • IPTG concentration: 0.2-0.5 mM

  • Induction time: 16-18 hours

• Sequence considerations: For partial LARS constructs, carefully select domain boundaries based on structural information to ensure proper folding. When expressing splice variants like LSV3, exact exon boundaries should be respected.

While specific expression conditions may require optimization for individual research objectives, these parameters provide a starting point based on successful expression of similar proteins.

What purification strategy yields the highest purity and activity for recombinant mouse LARS?

A multi-step purification strategy is recommended for obtaining high-purity, functionally active recombinant mouse LARS:

The purification protocol should include these important considerations:

• Include protease inhibitors in all buffers to prevent degradation

• Add 10% glycerol to storage buffers to maintain stability

• For maintaining enzymatic activity, include 5 mM MgCl₂ in the final buffer

• For catalytically active variants, verify activity using aminoacylation assays after purification

This approach typically yields protein with >95% purity suitable for most downstream applications including structural studies and functional assays .

How can the leucylation activity of recombinant mouse LARS be accurately measured?

The aminoacylation (leucylation) activity of recombinant mouse LARS can be measured using several complementary approaches:

Method 1: Radioactive aminoacylation assay

• Prepare reaction mixture containing:

  • Purified LARS (10-100 nM)

  • tRNA^Leu (2-5 μM)

  • [³H] or [¹⁴C]-labeled leucine (50-100 μM)

  • ATP (2 mM)

  • MgCl₂ (10 mM)

  • Buffer: 50 mM HEPES pH 7.5, 50 mM KCl

• Incubate at 37°C, taking aliquots at various time points

• Precipitate aminoacylated tRNA with TCA on filter paper

• Wash and measure radioactivity to quantify leucine attachment

Method 2: ATP-PPi exchange assay
This assay measures the reverse reaction catalyzed by LARS and is useful for assessing the first step of the aminoacylation reaction.

Method 3: Fluorescent-based assay
Using specially designed fluorescent substrates that change fluorescence properties upon aminoacylation.

For splice variants like LSV3, which lack catalytic activity, these assays can confirm the absence of leucylation capability while preserving the protein for subsequent signaling assays .

What methods are available to assess the mTOR signaling function of mouse LARS?

To evaluate the leucine-sensing and mTOR-activating functions of recombinant mouse LARS (including catalytically inactive variants like LSV3), researchers can employ the following methodological approaches:

Cell-based mTOR activation assays:

• Phosphorylation of mTOR targets:

  • Transfect cells with recombinant LARS or LSV3

  • Starve cells of leucine, then replenish

  • Measure phosphorylation of downstream mTOR targets (S6K, 4E-BP1) by Western blot

  • Compare activation kinetics between full-length LARS and LSV3

• Co-immunoprecipitation assays:

  • Express FLAG-tagged LARS or LSV3 in appropriate cell lines (e.g., HEK 293T)

  • Perform co-IP using FLAG antibodies

  • Analyze interactions with known partners (RagD, Vps34, other MSC components) by Western blot

  • This approach has confirmed that LSV3 maintains interactions with isoleucyl- and glutamyl-prolyl-tRNA synthetases, as well as signaling partners

• Leucine-binding assays:

  • Isothermal titration calorimetry (ITC) to measure direct leucine binding

  • Microscale thermophoresis (MST) to assess binding affinities

  • Compare binding parameters between wild-type LARS and splice variants

These complementary approaches allow comprehensive assessment of both the leucine-sensing capabilities and downstream signaling functions of mouse LARS variants .

How should experiments be designed to distinguish between the catalytic and signaling functions of mouse LARS?

Designing experiments to differentiate between the dual functions of mouse LARS requires careful consideration of experimental variables and appropriate controls. Here's a methodological framework:

Experimental design strategy:

• Use of catalytically inactive LARS variants:

  • Natural splice variants like LSV3 that lack aminoacylation activity but retain signaling functions

  • Engineered point mutations in catalytic residues

  • These enable isolation of signaling functions for independent study

• Factorial experimental design:

  • Complete factorial designs allow systematic evaluation of multiple variables and their interactions

  • For example, a 2×2×2 design might include factors such as:

    • LARS variant (full-length vs. LSV3)

    • Leucine concentration (low vs. high)

    • mTOR pathway inhibitor (present vs. absent)

  • This approach allows determination of main effects and interactions between factors

• Cell type considerations:

  • Use both immune cells (where LSV3 is naturally expressed) and non-immune cells

  • This comparison can reveal cell-type-specific regulation mechanisms

  • The alternative splicing of LARS is regulated by SRSF1 in a cell-type-specific manner

• Control design:

  • Empty vector controls

  • Catalytically inactive mutants for comparison with LSV3

  • Leucine-binding deficient mutants

This systematic approach enables researchers to parse the complex interplay between LARS's catalytic and signaling functions in different cellular contexts.

What statistical approaches are recommended for analyzing functional differences between LARS variants?

When investigating functional differences between mouse LARS variants, robust statistical analysis is essential. Based on experimental approaches in related studies, the following methodological framework is recommended:

For in vitro assays:

• Use at least triplicate measurements for each experimental condition

• Apply two-way ANOVA to analyze effects of both LARS variant and experimental conditions (e.g., leucine concentration)

• Include Bonferroni or Tukey post-hoc tests for multiple comparisons

For strain comparison studies:

• One-way ANCOVA using strain as an independent variable and body weight and sex as covariates

• For longitudinal studies, use two-way ANCOVA with appropriate covariates

• Post-hoc comparisons using the Bonferroni method when appropriate

For heritability analysis in genetic studies:

• Calculate heritability (h²) as the fraction of variance explained by strains in a simple ANOVA model

• Estimate locus effect size as the proportion of heritable variation explained by the peak marker at QTL regions

Power analysis considerations:

• For detecting differences between LARS variants with 80% power at α=0.05:

  • Small effect size (d=0.3): approximately 90 samples per group

  • Medium effect size (d=0.5): approximately 34 samples per group

  • Large effect size (d=0.8): approximately 14 samples per group

These statistical approaches ensure rigorous evaluation of functional differences while accounting for relevant biological variables .

How can mouse LARS be utilized in studying tissue-specific regulation of protein synthesis versus signaling?

The discovery of tissue-specific splice variants of LARS, particularly the leukocyte-specific LSV3, offers a powerful model for investigating differential regulation of translation and signaling pathways. Researchers can leverage this system through several methodological approaches:

• Tissue-specific expression profiling:

  • Quantify expression levels of LARS splice variants across different tissues using RT-qPCR

  • Correlate splice variant ratios with tissue-specific protein synthesis rates and mTOR activity

  • The leukocyte-specific exon skipping event in LARS provides a natural system to study tissue-specific regulation

• Regulatory mechanism investigation:

  • Examine the role of splicing factors like SRSF1 in regulating alternative splicing of LARS

  • Use CLIP-seq to identify direct RNA-protein interactions

  • Apply CRISPR-based approaches to modify splicing regulatory elements

  • SRSF1 has been shown to regulate LSV3 expression in a cell-type-specific manner

• Functional consequence analysis:

  • Compare protein synthesis rates and mTOR activity in tissues with different LARS splice variant profiles

  • Investigate how this balance affects cellular responses to nutrient availability

  • Examine implications for immune cell function, where LSV3 is predominantly expressed

• Developmental timing studies:

  • Track changes in LARS splicing during development and differentiation

  • Correlate with shifts in metabolic requirements and signaling pathway activity

This research direction could provide significant insights into how fundamental cellular processes are tailored to tissue-specific requirements through alternative splicing of multifunctional proteins like LARS .

How can genetic approaches like QTL mapping be applied to study LARS function in mouse models?

Quantitative trait locus (QTL) mapping offers powerful approaches for investigating the genetic basis of LARS function in complex physiological processes. Researchers can implement the following methodological framework:

• Experimental design for QTL studies:

  • Utilize BXD recombinant inbred mouse strains that show variable performance in LARS-dependent phenotypes

  • Phenotype mice for relevant traits (e.g., protein synthesis rates, mTOR signaling activity, response to leucine restriction)

  • Select strains with contrasting phenotypes to maximize genetic diversity and statistical power

• QTL mapping methodology:

  • Upload phenotypic data to GeneNetwork (www.genenetwork.org)

  • Apply fast linear regression equations of Haley and Knott to map QTLs

  • Compute likelihood ratio statistics (LRS) to assess genotype-phenotype associations

  • Perform permutation tests (2000 repetitions) to establish significance thresholds (p<0.63 for suggestive QTLs; p<0.05 for significant QTLs)

  • Calculate confidence intervals using 1.5 logarithm of the odds (LOD)

• Candidate gene analysis:

  • Evaluate genes within QTL confidence intervals based on:

    • Expression in relevant tissues (brain, skeletal muscle, immune cells)

    • Functional relevance from previous literature

    • Presence of polymorphisms (non-synonymous SNPs)

  • Assess expression, functional, and phenotypic information using MGI database and PubMed

  • Use GeneNetwork variant browser to identify SNP variants between parental haplotypes

• Validation experiments:

  • Confirm candidate gene effects using gene editing approaches (CRISPR/Cas9)

  • Perform rescue experiments with wildtype LARS or specific variants

  • Correlate molecular phenotypes with whole-organism traits

This comprehensive genetic approach can reveal novel insights into LARS function and regulation in complex physiological contexts, potentially identifying new therapeutic targets for LARS-related disorders .

What is the current understanding of how LARS splice variants may influence disease models?

The discovery that alternative splicing separates the catalytic and signaling functions of LARS has significant implications for disease models, particularly those involving immune function and metabolic regulation:

• Immunological disorders:

  • LSV3 is leukocyte-specific, suggesting specialized functions in immune cells

  • This splice variant lacks catalytic activity but maintains signaling functions, potentially allowing immune cells to regulate protein synthesis and mTOR signaling independently

  • This separation may be critical for balancing immune cell activation with metabolic demands

• Metabolic disease models:

  • LARS functions as a leucine sensor for mTOR, a master regulator of cellular metabolism

  • Different LARS variants may have distinct effects on metabolic sensing and subsequent cellular responses

  • Models of diabetes, obesity, and related metabolic disorders could be influenced by the balance of LARS variants

• Neurodevelopmental conditions:

  • BXD mouse strains show variability in motor coordination that may correlate with LARS function

  • Strains like BXD75 and BXD86 struggle with motor coordination and balance, possibly due to altered protein synthesis or mTOR signaling in neural tissues

  • These strains could serve as models for developmental coordination disorder (DCD) and related conditions

• Cancer models:

  • mTOR signaling is frequently dysregulated in cancer

  • The separation of LARS catalytic and signaling functions through alternative splicing may influence tumor metabolism and growth

  • Cancer cells might exploit specific LARS variants to maintain high translation rates while modulating mTOR signaling

Research investigating these disease connections is still emerging, but the unique properties of LARS splice variants provide promising avenues for understanding and potentially treating various pathological conditions .

What are the most promising approaches for studying the structural basis of LARS dual functionality?

Understanding the structural basis of LARS dual functionality represents a frontier in aminoacyl-tRNA synthetase research. Several methodological approaches show particular promise:

• Cryo-electron microscopy (cryo-EM) studies:

  • Visualize full-length LARS and splice variants like LSV3 in different functional states

  • Capture conformational changes upon leucine binding and during interactions with signaling partners

  • The 71-amino acid deletion in LSV3 affects residues approximately 30 Å apart in the LARS structure, suggesting significant structural rearrangements

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map conformational dynamics and solvent accessibility changes in different functional states

  • Compare flexibility and domain movements between full-length LARS and LSV3

  • Identify regions involved in allosteric communication between catalytic and signaling functions

• Integrative structural biology approaches:

  • Combine X-ray crystallography of individual domains with small-angle X-ray scattering (SAXS)

  • Use molecular dynamics simulations to model transitions between functional states

  • Validate models with cross-linking mass spectrometry and mutagenesis studies

• Structure-guided protein engineering:

  • Design minimal LARS constructs that retain either catalytic or signaling functions

  • Create chimeric proteins to probe domain contributions to specific functions

  • Develop biosensors based on LARS conformational changes for monitoring leucine levels or mTOR pathway activation

These approaches can provide unprecedented insights into how alternative splicing physically separates catalytic and signaling functions in this ancient and ubiquitous enzyme family .

How might comparative studies across species inform our understanding of LARS evolution and function?

Comparative studies of LARS across species offer valuable insights into the evolutionary trajectory of this ancient enzyme and its acquired signaling functions:

• Evolutionary analysis methodology:

  • Compare LARS sequences across diverse species, from bacteria to mammals

  • Construct phylogenetic trees to trace the emergence of signaling domains

  • Identify conserved and divergent regions in the catalytic core and peripheral domains

  • Exon 20, which is skipped in LSV3, contains some of the most conserved residues in LARS, highlighting the evolutionary significance of this alternative splicing event

• Functional conservation assessment:

  • Express and characterize LARS from multiple species

  • Compare catalytic parameters (kcat, KM) across evolutionary distance

  • Test signaling capabilities in reconstituted systems

  • Map the evolutionary acquisition of mTOR pathway interaction capabilities

• Alternative splicing conservation:

  • Survey LARS splice variants across vertebrate species

  • Determine whether the LSV3 splice variant is conserved in other mammals

  • Identify species-specific regulatory mechanisms for LARS splicing

  • The LSV3 leukocyte-specific exon skipping event may represent a mammalian-specific adaptation for immune function

• Correlation with physiological traits:

  • Link LARS structural features to species-specific metabolic requirements

  • Examine how variations in LARS structure correlate with differences in nutrient sensing and protein synthesis regulation

  • Investigate potential co-evolution with mTOR pathway components

This evolutionary perspective could reveal how nature has repurposed this ancient housekeeping enzyme for complex signaling functions, providing insights into both fundamental biology and potential therapeutic approaches .