Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.

Some researchers have previously shown that RNAi knockdown of arginyl-tRNA synthetase (ArgRS) before or after a hypoxic injury can rescue animals from death, based on the model organism, C. elegans. However, there has been no study on the application of arginyl-tRNA synthetase knockdown in treating mammalian ischemic stroke, and its potential mechanism and effect on ischemic brain damage are still unknown. Here, we focused on the Rars gene, which encodes an arginyl-tRNA synthetase, and examined the effects of Rars knockdown in a permanent middle cerebral artery occlusion model in rats. To achieve this aim, adult male Sprague-Dawley (SD) rats were given right cerebral cortex injections of short hairpin RNA (shRNA) adenovirus (AV) particles to knock down arginyl-tRNA synthetase, and a non-targeting control (NTC) vector or phosphate-buffered solution served as the controls. After 4 days, the rats were exposed to permanent middle cerebral artery occlusion (pMCAO). Then, the right cerebral cortex level of arginyl-tRNA synthetase was examined, and the effects of the Rars knockdown were evaluated by differences in infarction volume, oxidative stress, blood-brain barrier, mitochondrial function, and glucose metabolism at 1 day and 3 days after MCAO. The injection of shRNA adenovirus particles successfully suppressed the expression of arginyl-tRNA synthetase in the cerebral cortex. We observed an improvement in oxidative stress, mitochondrial function, and glucose utilization and a reduction in brain edema compared with the non-targeting control rats with suppressed expression of arginyl-tRNA synthetase mRNA in the ipsilateral ischemic cortex of the brain. Our findings indicate that knockdown of arginyl-tRNA synthetase in the cerebral cortex exerted neuroprotective effects, which were achieved not only by the improvement of oxidative stress and glucose utilization but also by the maintenance of mitochondrial morphological integrity and the preservation of mitochondrial function. Knockdown of ArgRS administration could be a promising approach to protect ischemic stroke.

Pelizaeus-Merzbacher disease (PMD) is a central nervous system (CNS) demyelinating disease in human, currently known as prototypic hypomyelinating leukodystrophy 1 (HLD1). The gene responsible for HLD1 encodes proteolipid protein 1 (PLP1), which is the major myelin protein produced by oligodendrocytes. HLD9 is an autosomal recessive disorder responsible for the gene differing from the plp1 gene. The hld9 gene encodes arginyl-tRNA synthetase (RARS), which belongs to a family of cytoplasmic aminoacyl-tRNA synthetases. Herein we show that HLD9-associated missense mutation of Ser456-to-Leu (S456L) localizes RARS proteins as aggregates into the lysosome but not into the endoplasmic reticulum (ER) and the Golgi body. In contrast, wild-type proteins indeed distribute throughout the cytoplasm. Expression of S456L mutant constructs in cells decreases lysosome-related signaling through ribosomal S6 protein phosphorylation, which is known to be required for myelin formation. Cells harboring the S456L mutant constructs fail to exhibit phenotypes with myelin web-like structures following differentiation in FBD-102b cells, as part of the mammalian oligodendroglial cell model, whereas parental cells exhibit them. Collectively, HLD9-associated RARS mutant proteins are specifically localized in the lysosome with downregulation of S6 phosphorylation involved in myelin formation, inhibiting differentiation in FBD-102b cells. These results present some of the molecular and cellular pathological mechanisms for defect in myelin formation underlying HLD9.

The expression changes of Rars gene in ischemia-injured neurons were investigated by detecting its translational product arginyl-tRNA synthetase (ArgRS), and the inhibitory effects of ischemic preconditioning (IPC) on Rars gene were explored. Both IPC model and prolonged ischemia (PI) model were established by using the classic oxygen glucose deprivation (OGD) method. The primary cultured neurons were assigned into the following groups: the experimental group (IPC+PI group), undergoing PI after a short period of IPC; the conditional control group (PI control group), subjected to PI without IPC; blank control group, the normally cultured neurons. The Rars transcriptional activities and ArgRS expression levels were measured at different time points after re-oxygenation (3 h/6 h/12 h/24 h). Data were collected and statistically analyzed. Compared to the blank control group, the Rars activities and ArgRS levels were significantly increased in PI control group, peaking at the time point of 6 h after re-oxygenation. Rars activities and ArgRS levels were significantly lower in the experimental group than in the PI control group at different time points after re-oxygenation. PI insult can induce an escalating activity of Rars and lead to ArgRS over-expression in primary cultured neurons. IPC can inhibit the increased Rars activity and down-regulate ArgRS expression of ischemia-insulted neurons. This mechanism may confer ischemic tolerance on neurons.

Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNA(Ser)UCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distinct from those possessed by the cytoplasmic tRNAArg in which the major identity elements permit the arginylation of all 5 encoded isoacceptors. We have performed comparative in vitro aminoacylation using an insect mitochondrial arginyl-tRNA synthetase and tRNAArgUCG structural variants. The established identity elements are sufficient to maintain the fidelity of tRNASerUCU reassignment. tRNAs having a UCU anticodon cannot be arginylated but can be converted to arginine acceptance by identity element transplantation. We have examined the evolutionary distribution and functionality of these tRNA elements within metazoan taxa. We conclude that the identity elements that have evolved for the recognition of mitochondrial tRNAArgUCG by the nuclear encoded mitochondrial arginyl-tRNA synthetases of eumetazoans have been extensively, but not universally conserved, throughout this clade. They ensure that the AGR codon reassignment in eumetazoan mitochondria is not compromised by misaminoacylation. In contrast, in other metazoans, such as Porifera, whose mitochondrial translation is dictated by the universal genetic code, recognition of the 2 encoded tRNAArgUCG/UCU isoacceptors is achieved through structural features that resemble those employed by the yeast cytoplasmic system.

In higher eukaryotes, one of the two arginyl-tRNA synthetases (ArgRSs) has evolved to have an extended N-terminal domain that plays a crucial role in protein synthesis and cell growth and in integration into the multisynthetase complex (MSC). Here, we report a crystal structure of the MSC subcomplex comprising ArgRS, glutaminyl-tRNA synthetase (GlnRS), and the auxiliary factor aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1)/p43. In this complex, the N-terminal domain of ArgRS forms a long coiled-coil structure with the N-terminal helix of AIMP1 and anchors the C-terminal core of GlnRS, thereby playing a central role in assembly of the three components. Mutation of AIMP1 destabilized the N-terminal helix of ArgRS and abrogated its catalytic activity. Mutation of the N-terminal helix of ArgRS liberated GlnRS, which is known to control cell death. This ternary complex was further anchored to AIMP2/p38 through interaction with AIMP1. These findings demonstrate the importance of interactions between the N-terminal domains of ArgRS and AIMP1 for the catalytic and noncatalytic activities of ArgRS and for the assembly of the higher-order MSC protein complex.

Arginyl-tRNA synthetase (ArgRS) is a tRNA-binding protein that catalyzes the esterification of L-arginine to its cognate tRNA. L-Canavanine, a structural analog of L-arginine, has recently been studied as an anticancer agent. Here, we determined the crystal structures of the apo, L-arginine-complexed, and L-canavanine-complexed forms of the cytoplasmic free isoform of human ArgRS (hArgRS). Similar interactions were formed upon binding to L-canavanine or L-arginine, but the interaction between Tyr312 and the oxygen of the oxyguanidino group was a little bit different. Detailed conformational changes that occur upon substrate binding were explained. The hArgRS structure was also compared with previously reported homologue structures. The results presented here may provide a basis for the design of new anticancer drugs, such as L-canavanine analogs.