AARS Human

What is the biochemical mechanism of human AARSs and how does it differ from prokaryotic counterparts?

The aminoacylation reaction catalyzed by human AARSs involves a two-step transesterification process. First, the AARS activates an amino acid with ATP to form an aminoacyl adenylate and pyrophosphate. Second, the amino acid residue is transferred from the adenylate conjugate onto the 3′-end of the tRNA, establishing an acyl ester linkage between the amino acid and the 3′-OH group in the ribose ring .

While the core catalytic function is conserved across all domains of life, human AARSs have acquired additional domains throughout evolution that are not present in bacterial enzymes. These appended domains are critical for non-canonical functions outside protein synthesis and increase the repertoire of interacting partners . Statistical analysis of domain conservation shows that pathogenic missense variants in human AARS proteins are significantly enriched in evolutionarily ancient domains, while benign variants are more commonly found in modern domains that emerged later in eukaryotes .

How are human AARSs organized structurally in cells?

In human cells, eight cytoplasmic AARSs associate with three non-synthetase proteins to form a large multi-tRNA synthetase complex (MSC). Cross-linking mass spectrometry (XL-MS) and molecular docking studies of the MSC in human HEK293T cells have revealed an unexpected asymmetric distribution of AARSs, featuring a clustering of tRNA anti-codon binding domains on one face of the complex .

This three-dimensional architecture provides structural information on flexibly appended domains, which are characteristic of nearly all MSC constituents. When investigating MSC organization, researchers should consider employing techniques like XL-MS that can capture information about these flexible domains that may not be resolved in crystallographic studies .

What is the relationship between AARS evolutionary history and human disease?

Research demonstrates a significant correlation between evolutionary conservation and disease pathogenicity in human AARSs. By leveraging evolutionary history data, researchers have established that pathogenic missense variants in human AARS proteins are enriched in evolutionarily ancient domains essential for the aminoacylation reaction, while benign or variants of unknown significance are more frequently found in modern domains that evolved more recently in eukaryotes .

This pattern suggests that mutations affecting the core catalytic functions have more severe consequences than those affecting newer domains. When assessing newly identified variants, researchers should consider the evolutionary context of the affected domain as part of their pathogenicity analysis. Methodologically, this requires determining the locations of modern and ancient domains in each AARS protein and statistically assessing the positional conservation across each domain .

What experimental approaches are most effective for investigating the dual roles of AARSs in translation and non-canonical functions?

To investigate both canonical and non-canonical functions of AARSs, researchers should implement a multi-faceted approach:

• Domain-specific mutation analysis: Engineer mutations that specifically affect either the catalytic domain or appended domains to dissect their respective contributions to different cellular processes .

• Interactome studies: Use techniques like co-immunoprecipitation followed by mass spectrometry to identify protein interaction partners specific to different AARS domains .

• Comparative functional assays: Design complementation assays in model organisms (like the yeast complementation systems mentioned in search result ) to assess the impact of variants on canonical function.

• Tissue-specific expression analysis: Given the tissue-specific manifestations of AARS-related disorders (particularly neurological), examine expression patterns in different tissues and cell types .

For studying non-canonical functions specifically, it's crucial to design controls that can distinguish effects resulting from impaired protein synthesis versus direct effects of the non-canonical function .

How should researchers approach the investigation of AARS mutations in neurological disorders?

Current evidence indicates that both cytosolic and mitochondrial AARSs are implicated in human nervous system pathologies, with at least 10 cytosolic and 14 mitochondrial AARSs linked to various neurological disorders . When investigating AARS mutations in neurological diseases, researchers should:

• Conduct comprehensive functional assays: Test variants using in vitro aminoacylation assays and in vivo complementation studies. For example, studies have shown that mutations like R618C in MARS failed to rescue the mes1Δ allele in yeast complementation assays, suggesting a functional deficit relevant to pathogenicity .

• Assess editing domain functionality: The case of the "sticky" mouse phenotype with the A734E mutation in the editing domain of cytosolic AlaRS demonstrates how compromised proofreading activity can lead to misfolded proteins and neurodegeneration. Researchers should evaluate both aminoacylation and editing functions when studying AARS variants .

• Examine tissue-specific effects: Initial assumptions that cytosolic AARS mutations primarily affect the peripheral nervous system while mitochondrial isoform mutations predominantly cause encephalopathies have proven overly simplistic. Research designs should consider both central and peripheral nervous system manifestations regardless of the subcellular localization of the AARS .

• Investigate protein misfolding mechanisms: Evidence suggests that AARS mutations can lead to neurodegeneration through global protein misfolding and overwhelming of the unfolded protein response, particularly in terminally differentiated neurons .

What methodologies are recommended for studying the three-dimensional architecture of the human multi-tRNA synthetase complex (MSC)?

The human MSC represents a challenging structural target due to its size and the presence of flexible domains. While partial or complete crystal structures of individual MSC constituents have been reported, the structure of the complete MSC remains elusive. Researchers investigating MSC architecture should consider:

• Cross-linking mass spectrometry (XL-MS): This technique has proven valuable for capturing structural information on flexibly appended domains that characterize nearly all MSC constituents .

• Integrative structural biology approaches: Combining data from multiple techniques including cryo-electron microscopy, small-angle X-ray scattering, and computational modeling with molecular docking .

• Functional domain mapping: Studies have revealed unexpected features such as the clustering of tRNA anti-codon binding domains on one face of the MSC, suggesting functional specialization that may be relevant to disease mechanisms .

When designing experiments, researchers should account for the asymmetric distribution of AARSs within the complex, as this may have functional implications not apparent from studies of individual components .

How do evolutionary constraints on AARS domains affect the interpretation of variant pathogenicity?

Recent research has established a framework for defining evolutionarily ancient and modern domains in AARS proteins using large, publicly accessible sequence databases . This evolutionary perspective offers valuable insights for variant interpretation:

• Domain-specific pathogenicity assessment: Statistical analyses demonstrate that pathogenic missense variants are significantly enriched in ancient domains, while benign variants are more common in modern domains. Researchers should weigh the evolutionary context of a variant's location when assessing its potential pathogenicity .

• Conservation scoring methodologies: Standardized approaches for determining ancient versus modern domains provide a reproducible framework that can be applied to any protein of interest, not just AARSs .

• Integrative analysis: For optimal variant classification, researchers should combine evolutionary domain analysis with functional studies, structural modeling, and clinical correlation .

This evolutionary approach to variant interpretation represents a powerful integration of evolutionary science, basic biochemistry, and clinical medicine that can improve diagnostic accuracy for individuals with AARS-related diseases .

What are the emerging methodologies for investigating disease mechanisms in AARS-related disorders?

Recent advances in understanding AARS-related disorders highlight several promising methodological approaches:

• Mouse models of editing domain mutations: The "sticky" mouse phenotype with A734E mutation in AlaRS's editing domain provides a valuable model for studying how compromised proofreading leads to protein misfolding and neurodegeneration. Similar approaches can be applied to other AARSs to investigate editing defects .

• Patient-derived cellular models: Creating induced pluripotent stem cells (iPSCs) from patients with AARS mutations and differentiating them into relevant neural cell types can provide insights into tissue-specific disease mechanisms .

• Protein quality control assessment: Given evidence that AARS mutations can lead to accumulation of misfolded proteins that overwhelm the unfolded protein response, methodologies for quantifying protein misfolding and quality control mechanisms are particularly valuable .

• Combinatorial functional approaches: Researchers should employ multiple complementary techniques to assess variant impact, including in vitro enzymatic assays, yeast complementation studies, and cell-based models of neurodegenerative processes .

These approaches reflect the current understanding that AARS-related disorders involve complex mechanisms beyond simple loss of aminoacylation function, including protein misfolding, disrupted cellular interactions, and potentially altered non-canonical functions .