Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy

Abstract

Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultra centrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to WT, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.

A recurrent GARS mutation causes distal hereditary motor neuropathy

“…Further, like most dominant GARS mutations that are associated with neuropathy (p.Glu125Gly, p.Pro152Leu, p.Leu183Pro, p.Asp200Asn, p.Asp215His, p.Ser265Phe, p.Leu272Gln, p.Pro298Leu, p.Glu333Gly, p.Ile334Phe, p.His472Arg, p.Gly580Arg, p.Gly652Ala), the p.Gly327Arg variant is not present in gnomAD (p.Gly294Arg has an allele count on 1). Because the p.Gly327Arg mutation affects a Gly residue that is conserved from yeast to mammals (Figure S1), we assessed the functional consequences of p.Gly327Arg mutation in a yeast complementation assay that has been previously used to test the functional consequences of disease‐associated ARS variants, including those identified in GARS . The p.Gly327Arg GARS variant did not support any yeast growth, indicating that this variant severely impairs protein function (Figure ).…”

“…Table 1 Gly327Arg mutation affects a Gly residue that is conserved from yeast to mammals ( Figure S1), we assessed the functional consequences of p.Gly327Arg mutation in a yeast complementation assay that has been previously used to test the functional consequences of disease-associated ARS variants, including those identified in GARS. 4,11 The p.Gly327Arg GARS variant did not support any yeast growth, indicating that this variant severely impairs protein function ( Figure 2). These data support the pathogenicity of the p.Gly327Arg…”

Peripheral neuropathy and cognitive impairment associated with a novel monoallelic HARS variant

“…Thirty‐seven different ARSs genes exist in humans. Among them, only three of them are functional in both cytoplasm and mitochondria, the rest acting exclusively either in the cytoplasm or in mitochondria . Mutations in 33 out of the 37 ARSs genes have been implicated in a large number of genetic disorders affecting multiple organs and tissues, including the nervous system .…”

“…In contrast, it is still unclear if peripheral neuropathies associated with dominant ARS mutations specifically affect the peripheral nervous system through loss‐of‐function, gain‐of‐function, or a combinatorial mechanism. Notably, the vast majority of neuropathy‐associated ARS alleles exhibit loss‐of‐function . The associated phenotypic spectrum is broad and encompasses both axonal and demyelinating motor and sensory neuropathies, as well as pure motor axonal neuropathy …”

“…The reason why mono‐allelic ARS mutations seem to affect preferentially the nervous system remains unclear, but may relate to the specialized process of local translation occurring in the distal axon. These ARSs‐neuropathy‐related genes are typically characterized by a decrease in aminoacylation activity when assessed in vivo and in vitro . Although some CMT mutations apparently have minimal effect on the activity of the mutant enzyme when assayed in vitro, protein synthesis measurements in the context of a model organism ( Drosophila melanogaster ) show a decrease nonetheless .…”