This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37°C for 20 min, with protein at 180 μg ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by widespread loss of lower motor neurons from the spinal cord. Lower motor neuron degeneration leads to a progressive decline in motor development, manifesting as muscle atrophy and weakness. It is now well characterised that ubiquitin homeostasis is altered in SMA and that reduction of the ubiquitin-like modifier-activating enzyme 1 (UBA1) is central to this disruption. UBA1 is responsible for activating ubiquitin as the first step in the ubiquitin conjugation process, marking unwanted proteins for degradation by the proteasome. While it is known that therapies targeting UBA1 rescue neuromuscular phenotypes in SMA models, the mechanism by which UBA1 mediates neurodegeneration is unclear. In fact, very little is known about the function of UBA1 beyond its canonical role in the ubiquitin proteasome system. To better understand the role of UBA1 in motor neuron degeneration, a robust set of antibodies for both in vivo and in vitro work to study UBA1 have been identified. This enabled a novel characterisation of UBA1 distribution throughout disease progression in SMA spinal motor neurons to be performed, revealing that UBA1 reduction is an important pre-symptomatic molecular feature of SMA. To identify downstream targets of UBA1 critical for UBA1-mediated degeneration in SMA, label-free proteomics was performed on HEK293 cells after overexpression or knockdown of UBA1. The proteomics data was analysed across multiple platforms, including Biolayout, IPA and DAVID to identify UBA1-dependent pathways and demonstrated that modulation of UBA1 levels lead to disruption of key cellular pathways including translation elongation, nuclear transport, and tRNA synthetases. Validation of target proteins from these UBA1-dependent pathways identified that the tRNA synthetease GARS behaves in a UBA1-dependent manner across a range of model systems in vitro and in vivo. It was then identified that GARS expression is significantly dysregulated across a range of neuronal tissues in a mouse model of SMA. Interestingly, mutations in GARS cause Charcot-Marie-Tooth disease type 2D (CMT2D), an axonal neuropathy, in which a disruption to sensory neuron fate in dorsal root ganglia has recently been identified. In a mouse model of SMA we identified a phenotype consistent with that in the CMT2D mouse model and showed that disruption to sensory neuron fate is reversible and dependent on changes in UBA1 and GARS expression in SMA. In conclusion, modulation of UBA1 levels leads to disruption of key cellular pathways, with dysregulation of tRNA synthetases a prominent feature that is likely to play a role in the pathogenesis of SMA.

No description available.