aminoacyl-tRNA Synthesis Research Articles

Potential Metabolite Biomarkers for Acute Versus Chronic Stage of Ischemic Stroke: A Pilot Study

Background: Metabolome profiling is used to identify biomarkers for acute ischemic stroke (AIS). Previous studies compared metabolite profiles in AIS and healthy controls, which did not account for factors that affect metabolome (genetics, medications). This pilot project evaluates the change in metabolite concentrations between the acute and chronic stage of stroke in the same cohort in order to minimize other factors impact. Methods: We performed global metabolome profile on serum of 20 and urine of 12 stroke patients in acute (72 hours) and chronic (3-5.2 months) stage and compared relative peak values using Wilcoxon and orthogonal partial least squares discriminant analysis methods. Chronic stage metabolite concentrations were considered baseline. We performed analysis to identify significantly overrepresented pathways using MetaboAnalyst. Results: Three serum metabolites asparagine (P = .045), tyrosine (P = .015), and xylose (P = .003) had significantly higher concentrations in acute stage. Seven out of top 10 serum metabolites ranked by Wilcoxon test P value were related to amino acid (AA) metabolism. Two urine metabolites glycine (P = .03) and acetylcarnitine (P = .05) had significantly different concentrations in the acute stage. Five of the top 10 urine metabolites related to AA metabolism. We identified 6 significant pathways after false discovery rate correction that were upregulated in the acute stage: (1) Aminoacyl-tRNA synthesis, (2) nitrogen, (3) alanine, aspartate, and glutamate, (4) branched-chain AA, (5) arginine and proline, and (6) phenylalanine metabolism. Conclusion: Longitudinal study design confirms that AA metabolism heavily involved in the pathophysiology of acute brain ischemia. Prospective longitudinal studies with a higher number of participants are needed to establish useful stroke biomarkers.

Trans-editing by aminoacyl-tRNA synthetase-like editing domains.

Aminoacyl-tRNA synthetases (aaRS) are ubiquitous enzymes responsible for aminoacyl-tRNA (aa-tRNA) synthesis. Correctly formed aa-tRNAs are necessary for proper decoding of mRNA and accurate protein synthesis. tRNAs possess specific nucleobases that promote selective recognition by cognate aaRSs. Selecting the cognate amino acid can be more challenging because all amino acids share the same peptide backbone and several are isosteric or have similar side chains. Thus, aaRSs can misactivate non-cognate amino acids and produce mischarged aa-tRNAs. If left uncorrected, mischarged aa-tRNAs deliver their non-cognate amino acid to the ribosome resulting in misincorporation into the nascent polypeptide chain. This changes the primary protein sequence and potentially causes misfolding or formation of non-functional proteins that impair cell survival. A variety of proofreading or editing pathways exist to prevent and correct mistakes in aa-tRNA formation. Editing may occur before the amino acid transfer step of aminoacylation via hydrolysis of the aminoacyl-adenylate. Alternatively, post-transfer editing, which occurs after the mischarged aa-tRNA is formed, may be carried out via a distinct editing site on the aaRS where the mischarged aa-tRNA is deacylated. In recent years, it has become clear that most organisms also encode factors that lack aminoacylation activity but resemble aaRS editing domains and function to clear mischarged aa-tRNAs in trans. This review focuses on these trans-editing factors, which are encoded in all three domains of life and function together with editing domains present within aaRSs to ensure that the accuracy of protein synthesis is sufficient for cell survival.

Quantitative proteomic analysis of xylose fermentation strain Pichia stipitis CBS 5776 to lignocellulosic inhibitors acetic acid, vanillin and 5-hydroxymethylfurfural.

To obtain a global insight into the dynamic protein expression pattern in Pichia stipitis during xylose fermentation in the presence of three representative inhibitors (acetic acid, vanillin and 5-hydroxymethylfurfural), proteins were extracted for quantitative proteomic analysis using 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) on a liquid chromatography-mass/mass spectrometry instrument. Interestingly, aconitase (Aco1p) and NAD-isocitrate dehydrogenase (Idh1p) were upregulated during the middle exponential phase in the presence of the three inhibitors during tricarboxylic acid cycle. We speculated that yeast cells adaptively increased the expression of the tricarboxylic acid cycle proteins to compensate for low NADH derived from glycolysis in the presence of the three inhibitors. Proteins related to amino acid metabolism, aminoacyl tRNA synthesis and stress response were also significantly affected in the presence of the three inhibitors. Taken together, quantitative proteomic analysis is capable of monitoring P. stipitis xylose fermentation under inhibitor conditions and identifying physiological changes, such as stress response.

PO-194 Urinary metabolomics study on effects of Rhodiola on Marathon Amateurs after Quantitative Exercise Load

Objective To study the effect of Chinese medicine Rhodiola on oxidative stress injury in amateur marathon runners after quantitative exercise load (20 km) and explore its mechanism.
 Methods Eight marathon amateurs were divided into four groups according to different test time, including before and after quantitative exercise load (group C and group CE), before and after quantitative exercise load after taking a month of the Rhodiola (group MC and group ME). The participants had serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and malondialdehyde (MDA), as well as myocardial enzyme index - creatine kinase (CK), lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB) and glutamic oxaline aminotransferase (AST/GOT) activity evaluated. In order to further explore the mechanism of action of Rhodiola, the urine was analyzed by 1H-HMR metabonomics technique.
 Results 1) Compared with group C, the activity of serum CK, LDH and AST/GOT of group CE increased significantly (P<0.01 or P<0.05), MDA content and SOD activity also increased significantly (P<0.05). A total of fifteen potential biomarkers were found in group CE, such as valine, lactic acid, 2- hydroxy isobutyric acid and so on (P<0.05 and VIP>1). 2) Compared with group CE, the activity of serum CK and AST/GOT and the content of MDA of group ME decreased significantly (P<0.05), eleven metabolites among the fifteen potential biomarkers reverted significantly (P<0.01 or P<0.05), which mainly involved in 4 metabolic pathways including alanine, aspartic acid and glutamic acid metabolism and so on.
 Conclusions Rhodiola can enhance the antioxidant capacity and improve myocardial damage of marathon amateurs after quantitative exercise load, which may be due to increased synthesis and utilization of aminoacyl-tRNA and other amino acids.

Quality control by trans-editing factor prevents global mistranslation of non-protein amino acid α-aminobutyrate

ABSTRACTAccuracy in protein biosynthesis is maintained through multiple pathways, with a critical checkpoint occurring at the tRNA aminoacylation step catalyzed by aminoacyl-tRNA synthetases (ARSs). In addition to the editing functions inherent to some synthetases, single-domain trans-editing factors, which are structurally homologous to ARS editing domains, have evolved as alternative mechanisms to correct mistakes in aminoacyl-tRNA synthesis. To date, ARS-like trans-editing domains have been shown to act on specific tRNAs that are mischarged with genetically encoded amino acids. However, structurally related non-protein amino acids are ubiquitous in cells and threaten the proteome. Here, we show that a previously uncharacterized homolog of the bacterial prolyl-tRNA synthetase (ProRS) editing domain edits a known ProRS aminoacylation error, Ala-tRNAPro, but displays even more robust editing of tRNAs misaminoacylated with the non-protein amino acid α-aminobutyrate (2-aminobutyrate, Abu) in vitro and in vivo. Our results indicate that editing by trans-editing domains such as ProXp-x studied here may offer advantages to cells, especially under environmental conditions where concentrations of non-protein amino acids may challenge the substrate specificity of ARSs.

Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.

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.

Determinants for tRNA-dependent pretransfer editing in the synthetic site of isoleucyl-tRNA synthetase.

The accurate expression of genetic information relies on the fidelity of amino acid–tRNA coupling by aminoacyl-tRNA synthetases (aaRS). When the specificity against structurally similar noncognate amino acids in the synthetic reaction does not support a threshold fidelity level for translation, the aaRS employ intrinsic hydrolytic editing to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of the finding that IleRS possessing an inactivated post-transfer editing domain is still capable of robust tRNA-dependent editing, we have recently proposed that the pretransfer editing activity resides within the synthetic site. Here we apply an improved methodology that allows quantitation of the AMP fraction that arises particularly from tRNA-dependent aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent pretransfer editing accounts for nearly one-third of the total proofreading by EcIleRS and that a highly conserved tyrosine within the synthetic site modulates both editing and aminoacylation. Therefore, synthesis of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping amino acid determinants. We suggest that this overlap hindered the evolution of synthetic site-based pretransfer editing as the predominant proofreading pathway, because that activity is difficult to accommodate in the context of efficient aminoacyl-tRNA synthesis. Instead, the acquisition of a spatially separate domain dedicated to post-transfer editing alone allowed for the development of a powerful deacylation machinery that effectively competes with dissociation of misacylated tRNAs.

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.

Coordinate Changes in Histone Modifications, mRNA Levels, and Metabolite Profiles in Clonal INS-1 832/13 β-Cells Accompany Functional Adaptations to Lipotoxicity

Lipotoxicity is a presumed pathogenetic process whereby elevated circulating and stored lipids in type 2 diabetes cause pancreatic β-cell failure. To resolve the underlying molecular mechanisms, we exposed clonal INS-1 832/13 β-cells to palmitate for 48 h. We observed elevated basal insulin secretion but impaired glucose-stimulated insulin secretion in palmitate-exposed cells. Glucose utilization was unchanged, palmitate oxidation was increased, and oxygen consumption was impaired. Halting exposure of the clonal INS-1 832/13 β-cells to palmitate largely recovered all of the lipid-induced functional changes. Metabolite profiling revealed profound but reversible increases in cellular lipids. Glucose-induced increases in tricarboxylic acid cycle intermediates were attenuated by exposure to palmitate. Analysis of gene expression by microarray showed increased expression of 982 genes and decreased expression of 1032 genes after exposure to palmitate. Increases were seen in pathways for steroid biosynthesis, cell cycle, fatty acid metabolism, DNA replication, and biosynthesis of unsaturated fatty acids; decreases occurred in the aminoacyl-tRNA synthesis pathway. The activity of histone-modifying enzymes and histone modifications of differentially expressed genes were reversibly altered upon exposure to palmitate. Thus, Insig1, Lss, Peci, Idi1, Hmgcs1, and Casr were subject to epigenetic regulation. Our analyses demonstrate that coordinate changes in histone modifications, mRNA levels, and metabolite profiles accompanied functional adaptations of clonal β-cells to lipotoxicity. It is highly likely that these changes are pathogenetic, accounting for loss of glucose responsiveness and perturbed insulin secretion.

A novel crystal form of pyrrolysyl-tRNA synthetase reveals the pre- and post-aminoacyl-tRNA synthesis conformational states of the adenylate and aminoacyl moieties and an asparagine residue in the catalytic site

Structures of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) have been determined in a novel crystal form. The triclinic form crystals contained two PylRS dimers (four monomer molecules) in the asymmetric unit, in which the two subunits in one dimer each bind N(ℇ)-(tert-butyloxycarbonyl)-L-lysyladenylate (BocLys-AMP) and the two subunits in the other dimer each bind AMP. The BocLys-AMP molecules adopt a curved conformation and the C(α) position of BocLys-AMP protrudes from the active site. The β7-β8 hairpin structures in the four PylRS molecules represent distinct conformations of different states of the aminoacyl-tRNA synthesis reaction. Tyr384, at the tip of the β7-β8 hairpin, moves from the edge to the inside of the active-site pocket and adopts multiple conformations in each state. Furthermore, a new crystal structure of the BocLys-AMPPNP-bound form is also reported. The bound BocLys adopts an unusually bent conformation, which differs from the previously reported structure. It is suggested that the present BocLys-AMPPNP-bound, BocLys-AMP-bound and AMP-bound complexes represent the initial binding of an amino acid (or pre-aminoacyl-AMP synthesis), pre-aminoacyl-tRNA synthesis and post-aminoacyl-tRNA synthesis states, respectively. The conformational changes of Asn346 that accompany the aminoacyl-tRNA synthesis reaction have been captured by X-ray crystallographic analyses. The orientation of the Asn346 side chain, which hydrogen-bonds to the carbonyl group of the amino-acid substrate, shifts by a maximum of 85-90° around the C(β) atom.

Taking AIM at the Start of Translation

Taking AIM at the Start of Translation

Trans-kingdom rescue of Gln-tRNA Gln synthesis in yeast cytoplasm and mitochondria

Aminoacylation of transfer RNAGln (tRNAGln) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNAGln is generated by direct glutaminylation of tRNAGln by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNAGln is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNAGln amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNAGln synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNAGln isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNAGln isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNAGln synthesis can be conferred by a single enzyme.

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA(fMet) and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structure-based alignment of the Rossmann fold identifies the insertion of an α-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.

Structural basis for specific aminoacyl-tRNA synthesis

Structural basis for specific aminoacyl-tRNA synthesis

Borrelidin, a small molecule nitrile-containing macrolide inhibitor of threonyl-tRNA synthetase, is a potent inducer of apoptosis in acute lymphoblastic leukemia

Due to the poor prognosis and limited therapeutic options for adult patients with acute lymphoblastic leukemia (ALL), development of novel therapies is much needed to prolong patient survival and increase the efficacy of their treatment. Malignant T cells need high levels of nutrients to maintain their proliferation rate. Borrelidin, a small molecule nitrile-containing macrolide, is an inhibitor of bacterial and eukaryal threonyl-tRNA synthetase. Borrelidin-mediated inhibition of aminoacyl-tRNA synthesis, leads to an induction in the levels of uncharged tRNA, nutritional stress and ultimately inhibition of protein synthesis. The aim of the present study was to investigate whether borrelidin treatment inhibits the proliferation of malignant ALL cell lines, Jurkat and CEM cells, and study the mechanism by which this drug acts. Our results show that borrelidin was able to potently inhibit the proliferation of ALL cell lines with a half maximal inhibitory concentration of 50ng/ml. Borrelidin showed a greater inhibitory effect on ALL cell lines compared to primary fibroblasts. Flow cytometry and western blot analysis indicated that borrelidin was able to increase the level of apoptosis and cause G(1) arrest in ALL cell lines. Activation of the general control nonderepressible-2 (GCN2) kinase stress responsive pathway and induction of CHOP protein was significantly higher in ALL cell lines treated with borrelidin. These findings collectively suggest for the first time that borrelidin targets ALL cell lines by inducing apoptosis and mediating G(1) arrest and that borrelidin treatment in ALL cell lines is correlated with activation of the GCN2 kinase pathway.

Heat Maps for Intramolecular Communication in an RNP Enzyme Encoding Glutamine

Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNA(Gln) complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 Å, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code.

Problem‐based test: An in vitro experiment to analyze the genetic code

Terms to be familiar with before you start to solve the test: genetic code, translation, synthetic polynucleotide, leucine, serine, filter precipitation, radioactivity measurement, template, mRNA, tRNA, rRNA, aminoacyl-tRNA synthesis, ribosomes, degeneration of the code, wobble, initiation, and elongation of protein synthesis, initiation codon.

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.

Codases: 50 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 the universal structural and functional characteristics of this largest family of enzymes, partitioned into two classes. The modes of tRNA binding and recognition, as well as additional editing activity, which are responsible for the extremely high fidelity of aminoacyl-tRNA synthesis, are discussed. The available data suggest an unusual evolutionary history for the most important components of the mechanism that ensures the proper synthesis of proteins and the association of this mechanism with amino acid biosynthesis. In addition, the review considers the secondary functions of synthetases in various cell metabolic processes, including pathophysiological ones. Their investigation may help to develop new diagnostic techniques and therapies.