Abstract
Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.
Highlights
Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders
After purification as initially reported for the WT enzyme[27], all were homogeneous according to dynamic light scattering (DLS) and SDS-PAGE criteria, with masses expected from their amino acid compositions
For both in vitro and in cellulo experiments, the mutants and the WT synthetase were prepared in parallel and treated in the same way for strictly comparative biochemical and biophysical analyses of solubility, thermal stability, 2D and 3D structure in solution
Summary
Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. Mitochondria are the powerhouses of the eukaryotic cell, hosting the production of energy in the form of ATP by oxidative phosphorylation of ADP They possess their own genome (mt-DNA), which codes in humans for 13 of the respiratory chain subunits, 22 tRNAs and 2 rRNAs. More than one thousand proteins, encoded by the nuclear genome, synthesized in the cytosol and subsequently imported into mitochondria, are required for mitochondrial biogenesis and functioning (reviewed in e.g.1). The cause of this selective vulnerability is not understood (reviewed in e.g.1,5,8,13), and neither is the way mutations affect the structure and/or function of mt-aaRSs. Human mt-AspRS is the most prominent case with the largest number of reported mutations (60). This leads to a frameshift and a premature stop, but the defect is called “leaky” because it does not fully stop the expression of full-length mt-AspRS6
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