Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Nematode asparaginyl-tRNA synthetase resolves intestinal inflammation in mice with T-cell transfer colitis.

The therapeutic effects of a controlled parasitic nematode infection on the course of inflammatory bowel disease (IBD) have been demonstrated in both animal and human models. However, the inability of individual well-characterized nematode proteins to recreate these beneficial effects has limited the application of component immunotherapy to human disease. The nematodes that cause chronic human lymphatic filariasis, Brugia malayi and Wuchereria bancrofti, are among the parasites that induce immune suppression. Filarial lymphatic pathology has been shown to involve NF-κB pathway-dependent production of vascular endothelial growth factor (VEGF), and stimulation of VEGF expression has also been reported by interleukin 8 (IL-8) via NF-κB pathways. Previously, we have shown that the filarial asparaginyl-tRNA synthetase (rBmAsnRS) interacts with IL-8 receptors using a combination of extracellular loops that differ from those bound by IL-8. To test the hypothesis that rBmAsnRS might induce an anti-inflammatory effect in vivo, we studied the effects of rBmAsnRS in an established murine colitis model using T-cell transfer mice. T-cell transfer colitis mice treated intraperitoneally with 100 µg of rBmAsnRS four times over 2 weeks showed resolution of cellular infiltration in the colonic mucosa, along with induction of a CD8(+) cellular response. In addition, rBmAsnRS induced a rise in IL-10 production from CD3(+) and lipopolysaccharide (LPS)- and cytosine phosphate guanosine (CPG)-stimulated splenic cells. In summary, this work demonstrates a novel anti-inflammatory nematode protein, supports the hygiene hypothesis, and supports continued refinement of alternative immunotherapies for treatment of IBD.

The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel.

The Helicobacter pylori (Hp) Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain-domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity of Hp AdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT's kinase activity (reduced by 55-75%) were identified. Mutations at Thr149 (37 Å distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT's kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.

Fibroblast growth factor 2-induced cytoplasmic asparaginyl-tRNA synthetase promotes survival of osteoblasts by regulating anti-apoptotic PI3K/Akt signaling.

Fibroblast growth factor 2 (FGF2), the potent bone anabolic agent, regulates the bone development, as well as the growth, remodeling and healing of the fracture. The intracellular signaling of FGF2 leads to activation of genes involved in cell proliferation, migration, differentiation and survival. However, little is known about FGF2-regulated proteins in the osteoblasts. Therefore, in this study, protein profiling in FGF2-treated MC3T3-E1 preosteoblast cells was evaluated using proteomic technologies. Six proteins including asparaginyl-tRNA synthetase (NARS), eukaryotic translation termination factor 1 (ETF1), GDP-forming succinyl-CoA synthetase (SUCLG2), heat shock protein 84 (HSP 84), sorting nexin 9 (SNX9) and alpha glucosidase 2alpha neutral subunit (GANAB) were increased more than 3-fold after the FGF2 treatment. Also, two proteins including beta-tropomyosin and tropomyosin 2 were decreased to 2-folds. Among these proteins, asparaginyl-tRNA synthetase (NARS), a member of aminoacyl-tRNA synthetases (AARS), was strikingly up-regulated more than 900-fold. The overexpression of NARS significantly increased the proliferation of both the MC3T3-E1 and the primary mouse calvarial cells. In contrast, significant reduction of the basal expression of NARS by siNARS remarkably suppressed the proliferation and induced the death of cell. After the siNARS treatment, the resistance to apoptosis induced by serum deprivation was also significantly reduced. The level of p-Akt was also reduced and the activity of caspase 3 significantly enhanced. In addition, NARS-induced protection against apoptosis was abolished by the treatment of PI3K inhibitors, wortmannin and LY294002. In conclusion, we suggest that NARS is one of the important mediators of FGF2 induced survival signaling in osteoblasts through the activation of PI3K/Akt survival pathway.

Systematic analysis of fusion and affinity tags using human aspartyl-tRNA synthetase expressed in E. coli.

Fusion and affinity tags are popular tools for the expression of mammalian proteins in bacteria. To facilitate the selection of expression approaches, a systematic comparison was performed. We cloned, sequenced, and expressed in Escherichia coli ubiquitin- and SUMO-hDRS fusion proteins with biotin- or 6xHis-tags. The tagging of hDRS with ubiquitin or SUMO was necessary to express properly folded and biologically active enzyme. Similar enhancement of hDRS activity was obtained by fusion to ubiquitin or SUMO. Ubiquitin, SUMO, biotin, and hexahistidine tags did not appreciably interfere with hDRS activity. Fusion proteins were specifically cleaved without altering the N-terminal of hDRS. After cleavage hDRS remained soluble and active with a specific activity comparable to that of the fused protein. Similar activity was observed with biotin- and 6xHis-tagging of hDRS. Higher purity but significantly lower yields of hDRS were obtained using biotin-tagging. Overall we demonstrated ubiquitin and SUMO fusion proteins similarly enhanced the proper folding of hDRS expressed in E. coli. In comparison to previous expressions of hDRS as a GST fusion, ubiquitin, and SUMO fusions provided higher yields and easier purification and cleavage.

Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase.

Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria.

Expression of human aspartyl-tRNA synthetase in COS cells.

Mammalian aspartyl-tRNA synthetase (DRS) occurs in a multi-enzyme complex of aminoacyl-tRNA synthetases, while DRS exists as free soluble enzymes in bacteria and yeast. The properties of human DRS transient expressed in COS cells were examined. After transfection of COS cells with the recombinant plasmids pSVL-63 that contained hDRS cDNA coding and non-coding sequences, and pSV-hDRS where the non-coding sequences were deleted, DRS in the transfected COS cells significantly increased compared to mock transfected cells. COS cells transfected with pSV-hDRS delta 32 that contained N-terminal 32 residue-coding sequence deleted hDRS cDNA showed no increase in DRS activity. Northern blot analysis showed that concentrations of corresponding mRNAs of hDRS and hDRS delta 32 were greatly enhanced in transfected cells. The increases in the level of the transcripts were much higher than those of the corresponding proteins. Gel filtration analysis showed that hDRS in pSV-hDRS transfected cells expressed as a low molecular weight form of hDRS and pSV-hDRS delta 32 transfected cells did not. Epitope tagging and indirect immunofluorescence microscopy was used to localize hDRS. Both hDRSmyc and hDRS delta 32myc were localized in the cytoplasm and showed diffused patterns. These results showed that hDRS has little tendency to aggregate in vivo and suggested that the N-terminal extension in hDRS was not involved in the expression and sub-cellular localization of hDRS, but may play a role in the maintenance of enzymatic activity of hDRS in COS cells.