Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites.

The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNA(Gln) and a glutaminyl-tRNA amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln). Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyl-tRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNA(Gln) to Gln-tRNA(Gln) in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria.

Kinetics of precursor labeling in stable isotope labeling in cell cultures (SILAC) experiments.

Recent advances in mass spectrometry have enabled proteome-wide analyses of cellular protein turnover. These studies have been greatly propelled by the development of stable isotope labeling in cell cultures (SILAC), a set of standardized protocols, reagents aimed at quantifying the incorporation of (15)N/(13)C labeled amino acids into proteins. In dynamic SILAC experiments, the degree of isotope incorporation in proteins is measured over time and used to determine turnover kinetics. However, the kinetics of isotope incorporation in proteins can potentially be influenced not only by their intracellular turnover but also by amino acid uptake, recycling and aminoacyl-tRNA synthesis. To assess the influence of these processes in dynamic SILAC experiments, we have measured the kinetics of isotopic enrichment within intracellular free amino acid and aminoacyl-tRNA precursor pools in dividing and division-arrested neuroblastoma cells following the introduction of extracellular (15)N labeled amino acids. We show that the total flux of extracellular amino acids into cells greatly exceeds that of intracellular amino acid recycling and synthesis. Furthermore, in comparison to internal sources, external amino acids are preferentially utilized as substrates for aminoacyl-tRNA precursors for protein synthesis. As a result, in dynamic SILAC experiments conducted in culture, the aminoacyl-tRNA precursor pool is near completely labeled in a few hours and protein turnover is the limiting factor in establishing the labeling kinetics of most proteins.

Quality control and infiltration of translation by amino acids outside of the genetic code.

Translation of the genome into functional proteins is critical for cellular life. Accurate protein synthesis relies on proper decoding of mRNAs by the ribosome using aminoacyl-tRNAs. During aminoacyl-tRNA synthesis, stringent substrate discrimination and rigorous product proofreading ensure tRNAs are paired with the correct amino acid, as defined by the rules of the genetic code. What has remained far less clear is the extent to which amino acids that are not part of the genetic code might also threaten translational accuracy. Here, we review the broad range of nonproteinogenic, or nonprotein, amino acids that can naturally accumulate under different conditions, the ability of the translation quality control machinery to deal with such substrates, and their potential impact on the integrity of the genetic code and cellular viability.

Synthetic and editing mechanisms of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRS) ensure the faithful transmission of genetic information in all living cells. The 24 known aaRS families are divided into 2 structurally distinct classes (class I and class II), each featuring a catalytic domain with a common fold that binds ATP, amino acid, and the 3'-terminus of tRNA. In a common two-step reaction, each aaRS first uses the energy stored in ATP to synthesize an activated aminoacyl adenylate intermediate. In the second step, either the 2'- or 3'-hydroxyl oxygen atom of the 3'-A76 tRNA nucleotide functions as a nucleophile in synthesis of aminoacyl-tRNA. Ten of the 24 aaRS families are unable to distinguish cognate from noncognate amino acids in the synthetic reactions alone. These enzymes possess additional editing activities for hydrolysis of misactivated amino acids and misacylated tRNAs, with clearance of the latter species accomplished in spatially separate post-transfer editing domains. A distinct class of trans-acting proteins that are homologous to class II editing domains also perform hydrolytic editing of some misacylated tRNAs. Here we review essential themes in catalysis with a view toward integrating the kinetic, stereochemical, and structural mechanisms of the enzymes. Although the aaRS have now been the subject of investigation for many decades, it will be seen that a significant number of questions regarding fundamental catalytic functioning still remain unresolved.

Essential nontranslational functions of tRNA synthetases.

Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. Although these new functions were thought to be 'moonlighting activities', many are as critical for cellular homeostasis as their activity in translation. New roles have been associated with their cytoplasmic forms as well as with nuclear and secreted extracellular forms that affect pathways for cardiovascular development and the immune response and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. New architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. Although a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid-binding site for another purpose.

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.

Riboswitch (T-box)-mediated control of tRNA-dependent amidation in Clostridium acetobutylicum rationalizes gene and pathway redundancy for asparagine and asparaginyl-trnaasn synthesis.

Analysis of the Gram-positive Clostridium acetobutylicum genome reveals an inexplicable level of redundancy for the genes putatively involved in asparagine (Asn) and Asn-tRNA(Asn) synthesis. Besides a duplicated set of gatCAB tRNA-dependent amidotransferase genes, there is a triplication of aspartyl-tRNA synthetase genes and a duplication of asparagine synthetase B genes. This genomic landscape leads to the suspicion of the incoherent simultaneous use of the direct and indirect pathways of Asn and Asn-tRNA(Asn) formation. Through a combination of biochemical and genetic approaches, we show that C. acetobutylicum forms Asn and Asn-tRNA(Asn) by tRNA-dependent amidation. We demonstrate that an entire transamidation pathway composed of aspartyl-tRNA synthetase and one set of GatCAB genes is organized as an operon under the control of a tRNA(Asn)-dependent T-box riboswitch. Finally, our results suggest that this exceptional gene redundancy might be interconnected to control tRNA-dependent Asn synthesis, which in turn might be involved in controlling the metabolic switch from acidogenesis to solventogenesis in C. acetobutylicum.

Microcin C: biosynthesis and mechanisms of bacterial resistance.

Nonhydrolyzable aminoacyl-adenylates that inhibit protein synthesis provide a promising route towards the development of novel antibiotics whose mechanism of action limits the appearance of bacterial drug resistance. The 'Trojan horse' antibiotic microcin C (McC) consists of a nonhydrolyzable aspartyl-adenylate that is efficiently imported into bacterial cells owing to a covalently attached peptide carrier. Once inside the cell, the carrier is removed by proteolytic processing to release a potent aspartyl tRNA synthetase inhibitor. The focus of this article is on the mechanism of biosynthesis of McC. We also examine the strategies utilized by McC-producing strains to overcome toxicity due to unwanted, premature processing of the drug. This article will discuss how McC biosynthesis can be systematically manipulated for the development of derivatives that will target the entire battery of aminoacyl tRNA synthetases in various bacteria.

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.

Quality control in aminoacyl-tRNA synthesis its role in translational fidelity.

Accurate translation of mRNA into protein is vital for maintenance of cellular integrity. Translational fidelity is achieved by two key events: synthesis of correctly paired aminoacyl-tRNAs by aminoacyl-tRNA synthetases (aaRSs) and stringent selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. AaRSs define the genetic code by catalyzing the formation of precise aminoacyl ester-linked tRNAs via a two-step reaction. AaRSs ensure faithful aa-tRNA synthesis via high substrate selectivity and/or by proofreading (editing) of noncognate products. About half of the aaRSs rely on proofreading mechanisms to achieve high levels of accuracy in aminoacylation. Editing functions in aaRSs contribute to the overall low error rate in protein synthesis. Over 40 years of research on aaRSs using structural, biochemical, and kinetic approaches has expanded our knowledge of their cellular roles and quality control mechanisms. Here, we review aaRS editing with an emphasis on the mechanistic and kinetic details of the process.

Identification and categorization of liver toxicity markers induced by a related pair of drugs.

Drug-induced liver injury (DILI) is the primary adverse event that results in the withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in the pre-clinical or clinical phases of drug development. This paper presents an approach for identifying potential liver toxicity genomic biomarkers from a liver toxicity biomarker study involving the paired compounds entacapone ("non-liver toxic drug") and tolcapone ("hepatotoxic drug"). Molecular analysis of the rat liver and plasma samples, combined with statistical analysis, revealed many similarities and differences between the in vivo biochemical effects of the two drugs. Six hundred and ninety-five genes and 61 pathways were selected based on the classification scheme. Of the 61 pathways, 5 were specific to treatment with tolcapone. Two of the 12 animals in the tolcapone group were found to have high ALT, AST, or TBIL levels. The gene Vars2 (valyl-tRNA synthetase 2) was identified in both animals and the pathway to which it belongs, the aminoacyl-tRNA biosynthesis pathway, was one of the three most significant tolcapone-specific pathways identified.

Synthesis of Glu-tRNA(Gln) by engineered and natural aminoacyl-tRNA synthetases.

A protein engineering approach to delineating which distinct elements of homologous tRNA synthetase architectures are responsible for divergent RNA-amino acid pairing specificities is described. Previously, we constructed a hybrid enzyme in which 23 amino acids from the catalytic domain of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) were replaced with the corresponding residues of human glutamyl-tRNA synthetase (GluRS). The engineered hybrid (GlnRS S1/L1/L2) synthesizes Glu-tRNA(Gln) more than 10(4)-fold more efficiently than GlnRS. Detailed comparison of kinetic parameters between GlnRS S1/L1/L2 and the naturally occurring Methanothermobacter thermautotrophicus GluRS(ND), which is also capable of Glu-tRNA(Gln) synthesis, now shows that both k(cat) and K(m) for glutamate are recapitulated in the engineered enzyme, but that K(m) for tRNA is 200-fold higher. Thus, the simultaneous optimization of paired amino acid and tRNA binding sites found in a naturally occurring enzyme is not recapitulated in a hybrid that is successfully engineered for amino acid complementarity. We infer that the GlnRS architecture has differentiated to match only cognate amino acid-RNA pairs, and that the substrate selection functions do not operate independently of each other. Design and characterization of four additional hybrids identify further residues involved in improving complementarity for glutamate and in communicating between amino acid and tRNA binding sites. The robust catalytic function demonstrated in this engineered system offers a novel platform for exploring the stereochemical origins of coding as a property of the ancient Rossmann fold.

tRNA as an active chemical scaffold for diverse chemical transformations.

During protein synthesis, tRNA serves as the intermediary between cognate amino acids and their corresponding RNA trinucleotide codons. Aminoacyl-tRNA is also a biosynthetic precursor and amino acid donor for other macromolecules. AA-tRNAs allow transformations of acidic amino acids into their amide-containing counterparts, and seryl-tRNA(Ser) donates serine for antibiotic synthesis. Aminoacyl-tRNA is also used to cross-link peptidoglycan, to lysinylate the lipid bilayer, and to allow proteolytic turnover via the N-end rule. These alternative functions may signal the use of RNA in early evolution as both a biological scaffold and a catalyst to achieve a wide variety of chemical transformations.

Biogenesis of glutaminyl-mt tRNAGln in human mitochondria.

Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA(Gln) is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA(Gln) to form Glu-tRNA(Gln). In addition, we have identified a human homolog of the Glu-tRNA(Gln) amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA(Gln) accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA(Gln) formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA(Gln) has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA(Gln) is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.

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.

[Codases: fifty years after].

Aminoacyl-tRNA synthetases (codases) catalyze aminoacylation of a particular tRNA with the corresponding amino acid at the first step of protein biosynthesis. The review considers universal structure-functional characteristics of the largest family of enzymes partitioned into two classes. Modes of tRNA binding and recognition, and additional editing activity, which are responsible for the fidelity of aminoacyl-tRNA synthesis, are discussed. The aaRSs catalytic cores are highly relevant to the ancient metabolic reactions, namely, amino acids and cofactors biosynthesis. Thus, the biosynthetic machinery for producing amino acids had a profound effect on almost every aspect of aminoacylation reaction. The review also deals with secondary functions of synthetases in various processes of cell metabolism. Certain of these functions have to do with complex pathophysiological mechanisms involved in disease production. Their investigation may help to develop new diagnostic techniques and therapies.

Aminoacyl-tRNA synthesis and translational quality control.

Translating the 4-letter code of RNA into the 22-letter alphabet of proteins is a central feature of cellular life. The fidelity with which mRNA is translated during protein synthesis is determined by two factors: the availability of aminoacyl-tRNAs composed of cognate amino acid:tRNA pairs and the accurate selection of aminoacyl-tRNAs on the ribosome. The role of aminoacyl-tRNA synthetases in translation is to define the genetic code by accurately pairing cognate tRNAs with their corresponding amino acids. Synthetases achieve the amino acid substrate specificity necessary to keep errors in translation to an acceptable level in two ways: preferential binding of the cognate amino acid and selective editing of near-cognate amino acids. Editing significantly decreases the frequency of errors and is important for translational quality control, and many details of the various editing mechanisms and their effect on different cellular systems are now starting to emerge.

Redundant synthesis of cysteinyl-tRNACys in Methanosarcina mazei.

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants.

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA(Gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA(Gln) is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA(Gln), which is the only tRNA(Gln) present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA(Gln) formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.