CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS

Abstract

Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the pathology.

A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations

“…Although there has been debate (32, 51) as to whether AARS1 functions as a dimer or a monomer, the results of our study demonstrate that it likely functions as a dimer in vivo , which is a requirement for a dominant-negative mechanism of disease. Previous studies using purified AARS1 protein for in vitro enzymatic assays determined that the C-terminal dimerization domain (beginning at G757) is dispensable for aminoacylation (51).…”

“…While the data presented here strongly argue in favor of a dominant-negative effect, it is possible that a dominant-negative and neomorphic gain-of-function effects work in concert to exacerbate neuronal pathology. A recent study showed that the pathogenic AARS1 variants N71Y, G102R, and R329H each cause a conformational change that enables binding to Neuropilin-1 (32), a widely expressed receptor that modulates a variety of signalling pathways and that is critical for neurovascular development (57). Although such an interaction might compound the damage in patient neurons, our yeast model demonstrates that a neuron-specific (or mammalian-specific) interaction is not required for pathogenic AARS1 alleles to exert dominant effects.…”

“…It is currently unknown if the dozens of identified pathogenic variants across the five implicated ARS genes (10,(21)(22)(23)(24)(25)(26)(27)(28) exert a similar effect on a shared pathway, or if there are unique pathogenic mechanisms for each locus. One proposed mechanism is a neo-morphic gain-of-function effect, in which mutations expose novel protein interfaces that facilitate aberrant protein-protein interactions, leading to dysregulated neuronal pathways (29)(30)(31)(32)(33). However, it is unlikely that the dozens of different alleles across all five ARS loci give rise to an identical gain-of-function effect via aberrant interactions.…”

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

“…The necessity of GlyRS binding NRP1 for the disease mechanism leading to neuropathy is called into question by the failure of NRP1 to bind ΔETAQ, a four amino acid internal deletion in GlyRS that causes a severe, early onset neuropathy in both mice and humans ( Morelli et al, 2019 ). Nonetheless, several other neuropathy-associated mutant GlyRS proteins bind NRP1, as do CMT2N-associated alleles of AARS , suggesting that it may in some way be contributing to the disease severity and pathogenesis ( He et al, 2015 ; Sun et al, 2021 ). This also suggests that small molecules to block this interaction or antibodies to clear mutant GlyRS proteins could be therapeutic strategies.…”

RNA Polymerase III Subunit Mutations in Genetic Diseases

“…Dominant mutations in several genes encoding tRNA aminoacyl synthetases are associated with Charcot-Marie-Tooth (CMT) disease and characterization of the corresponding mutants has demonstrated that aminoacylation activity is frequently not impaired. Instead, the mutations induce an alternative open conformation of the enzyme, which exposes a surface for new protein interactions (He et al, 2011;Blocquel et al, 2017;Bervoets et al, 2019;Blocquel et al, 2019;Sun et al, 2021), indicating a gain-of-function mechanism. In the case of leukodystrophies caused by bi-allelic mutations in genes encoding aaRS1, caused by hypomorphic mutations, further studies are required to determine if there is an underlying mechanism that involves translation deregulation and/or shares features with POLR3-HLD.…”