Resilience and proteome response of Escherichia coli to high levels of isoleucine mistranslation.

Accurate pairing of amino acids and tRNAs is a prerequisite for faithful translation of genetic information during protein biosynthesis. Here we present the effects of proteome-wide mistranslation of isoleucine (Ile) by canonical valine (Val) or non-proteinogenic norvaline (Nva) in a genetically engineered Escherichia coli strain with an editing-defective isoleucyl-tRNA synthetase (IleRS). Editing-defective IleRS efficiently mischarges both Val and Nva to tRNAIle and impairs the translational accuracy of Ile decoding. When mistranslation was induced by the addition of Val or Nva to the growth medium, an Ile-to-Val or Ile-to-Nva substitution of up to 20 % was measured by high-resolution mass spectrometry. This mistranslation level impaired bacterial growth, promoted the SOS response and filamentation during stationary phase, caused global proteome dysregulation and upregulation of the cellular apparatus for maintaining proteostasis, including the major chaperones (GroES/EL, DnaK/DnaJ/GrpE and HtpG), the disaggregase ClpB and the proteases (Lon, HslV/HslU, ClpA, ClpS). The most important consequence of mistranslation appears to be non-specific protein aggregation, which is effectively counteracted by the disaggregase ClpB. Our data show that E. coli can sustain high isoleucine mistranslation levels and remain viable despite excessive protein aggregation and severely impaired translational fidelity. However, we show that inaccurate translation lowers bacterial resilience to heat stress and decreases bacterial survival at elevated temperatures.

In search of new stratification strategies: tissue proteomic profiling of papillary thyroid microcarcinoma in patients with localized disease and lateral neck metastases.

INTRODUCTION: Papillary thyroid carcinomas (PTC) are the most common thyroid malignancies that are often diagnosed as microcarcinomas when the tumor is less than one centimetre in diameter. Currently, there are no valid stratification strategies that would reliably assess the risk of lateral neck metastases and optimize surgical treatment. MATERIALS AND METHODS: Aiming to find potential tissue biomarkers of metastatic potential, we conducted a cross-sectional proteomic pilot study on formalin-fixed paraffin-embedded tissues of metastatic (N = 10) and non-metastatic (N = 10) papillary thyroid microcarcinoma patients. Samples were analysed individually using liquid chromatography/mass spectrometry, and the differentially expressed proteins (DEP) were functionally annotated. RESULTS: We identified five overexpressed DEPs in the metastatic group (EPB41L2, CSE1L, GLIPR2, FGA and FGG) with a known association to tumour biology. Using bioinformatic-based tools, we found markedly different profiles of significantly enriched biological processes between the two groups. CONCLUSIONS: The identified DEPs might have a role as potential tissue biomarkers for PTC metastases. However, further prospective research is needed to confirm our findings.

Evolutionarily conserved cysteines in plant cytosolic seryl-tRNA synthetase are important for its resistance to oxidation.

We have previously identified a unique disulfide bond in the crystal structure of Arabidopsis cytosolic seryl-tRNA synthetase involving cysteines evolutionarily conserved in all green plants. Here, we discovered that both cysteines are important for protein stability, but with opposite effects, and that their microenvironment may promote disulfide bond formation in oxidizing conditions. The crystal structure of the C244S mutant exhibited higher rigidity and an extensive network of noncovalent interactions correlating with its higher thermal stability. The activity of the wild-type showed resistance to oxidation with H2 O2 , while the activities of cysteine-to-serine mutants were impaired, indicating that the disulfide link may enable the protein to function under oxidative stress conditions which can be beneficial for an efficient plant stress response.

Stage II of Chronic Kidney Disease-A Tipping Point in Disease Progression?

Chronic kidney disease (CKD) is the progressive loss of renal function. Although advances have been made in understanding the progression of CKD, key molecular events in complex pathophysiological mechanisms that mark each stage of renal failure remain largely unknown. Changes in plasma protein profiles in different disease stages are important for identification of early diagnostic markers and potential therapeutic targets. The goal of this study was to determine the molecular profile of each CKD stage (from 1 to 5), aiming to specifically point out markedly expressed or downregulated proteins. We performed a cross-sectional shotgun-proteomic study of pooled plasma across CKD stages and compared them to healthy controls. After sample pooling and heparin-column purification we analysed proteomes from healthy to CKD stage 1 through 5 participants' plasma by liquid-chromatography/mass-spectrometry. We identified 453 proteins across all study groups. Our results indicate that key events, which may later affect the course of disease progression and the overall pathophysiological background, are most pronounced in CKD stage 2, with an emphasis on inflammation, lipoprotein metabolism, angiogenesis and tissue regeneration. We hypothesize that CKD stage 2 is the tipping point in disease progression and a suitable point in disease course for the development of therapeutic solutions.

A pair of isoleucyl-tRNA synthetases in Bacilli fulfills complementary roles to keep fast translation and provide antibiotic resistance.

Isoleucyl-tRNA synthetase (IleRS) is an essential enzyme that covalently couples isoleucine to the corresponding tRNA. Bacterial IleRSs group in two clades, ileS1 and ileS2, the latter bringing resistance to the natural antibiotic mupirocin. Generally, bacteria rely on either ileS1 or ileS2 as a standalone housekeeping gene. However, we have found an exception by noticing that Bacillus species with genomic ileS2 consistently also keep ileS1, which appears mandatory in the family Bacillaceae. Taking Priestia (Bacillus) megaterium as a model organism, we showed that PmIleRS1 is constitutively expressed, while PmIleRS2 is stress-induced. Both enzymes share the same level of the aminoacylation accuracy. Yet, PmIleRS1 exhibited a two-fold faster aminoacylation turnover (kcat ) than PmIleRS2 and permitted a notably faster cell-free translation. At the same time, PmIleRS2 displayed a 104 -fold increase in its Ki for mupirocin, arguing that the aminoacylation turnover in IleRS2 could have been traded-off for antibiotic resistance. As expected, a P. megaterium strain deleted for ileS2 was mupirocin-sensitive. Interestingly, an attempt to construct a mupirocin-resistant strain lacking ileS1, a solution not found among species of the family Bacillaceae in nature, led to a viable but compromised strain. Our data suggest that PmIleRS1 is kept to promote fast translation, whereas PmIleRS2 is maintained to provide antibiotic resistance when needed. This is consistent with an emerging picture in which fast-growing organisms predominantly use IleRS1 for competitive survival.

The MATH-BTB Protein TaMAB2 Accumulates in Ubiquitin-Containing Foci and Interacts With the Translation Initiation Machinery in Arabidopsis.

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin-dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

Synthesis and In Vitro Screening of Novel Heterocyclic ß-d-Gluco- and ß-d-Galactoconjugates as Butyrylcholinesterase Inhibitors.

The development of selective butyrylcholinesterase (BChE) inhibitors may improve the treatment of Alzheimer's disease by increasing lower synaptic levels of the neurotransmitter acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and improved cognitive functions. A series of 14 novel heterocyclic ß-d-gluco- and ß-d-galactoconjugates were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and 1-benzylbenzimidazolium substituted ß-d-gluco- and ß-d-galacto-derivatives in a 10-50 micromolar range. The analysis performed by molecular modelling indicated key residues of the BChE active site, which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor should enable better blood-brain barrier permeability, and thus increase bioavailability in the central nervous system of these compounds.

On the Mechanism and Origin of Isoleucyl-tRNA Synthetase Editing against Norvaline.

Aminoacyl-tRNA synthetases (aaRSs), the enzymes responsible for coupling tRNAs to their cognate amino acids, minimize translational errors by intrinsic hydrolytic editing. Here, we compared norvaline (Nva), a linear amino acid not coded for protein synthesis, to the proteinogenic, branched valine (Val) in their propensity to mistranslate isoleucine (Ile) in proteins. We show that in the synthetic site of isoleucyl-tRNA synthetase (IleRS), Nva and Val are activated and transferred to tRNA at similar rates. The efficiency of the synthetic site in pre-transfer editing of Nva and Val also appears to be similar. Post-transfer editing was, however, more rapid with Nva and consequently IleRS misaminoacylates Nva-tRNAIle at slower rate than Val-tRNAIle. Accordingly, an Escherichia coli strain lacking IleRS post-transfer editing misincorporated Nva and Val in the proteome to a similar extent and at the same Ile positions. However, Nva mistranslation inflicted higher toxicity than Val, in agreement with IleRS editing being optimized for hydrolysis of Nva-tRNAIle. Furthermore, we found that the evolutionary-related IleRS, leucyl- and valyl-tRNA synthetases (I/L/VRSs), all efficiently hydrolyze Nva-tRNAs even when editing of Nva seems redundant. We thus hypothesize that editing of Nva-tRNAs had already existed in the last common ancestor of I/L/VRSs, and that the editing domain of I/L/VRSs had primarily evolved to prevent infiltration of Nva into modern proteins.

Susceptible and protective HLA-DR and HLA-DQ alleles for Fya alloimmunization in the Croatian population.

BACKGROUND: Alloimmunization is a known risk of transfusion therapy caused by exposure to foreign RBC antigens. However, alloimmunization is not observed in all transfused patients. Human leukocyte antigen (HLA) molecules may contribute to the recognition and presentation of foreign antigens and to the potency of immune responses that result in the production of antibodies. The aim of this study was to determine the association of HLA-DR and HLA-DQ polymorphisms with alloimunization to Fya antigen in Croatian patients. STUDY DESIGN AND METHODS: The study was conducted on 70 alloimmunized patients to Fya antigen and two control groups: 165 healthy Croatian individuals (Control 1) and 45 Fya antigen-negative nonimmunized patients exposed to Fya antigen (Control 2). Phenotype frequencies for HLA-DRB1 and HLA-DQB1 alleles were compared between the cases and control groups. RESULTS: Statistically significant differences in phenotype frequencies between cases and controls were found for DRB1*04 (odds ratios [ORs], 10.5 and 18.7 for Control 1 and Control 2, respectively), DRB1*15 (ORs, 8.0 and 6.9), and DQB1*02 alleles (ORs, 0.2 and 0.03); and DRB1*04-DQB1*03:01 (ORs, 7.9 and 17.6), DRB1*04-DQB1*03:02 (ORs, 5.5 and 7.6), DRB1*15-DQB1*06:02 (ORs, 7.3 and 5.5), DRB1*03-DQB1*02:01 (OR, 0.1), and DRB1*07-DQB1*02:02 (OR, 0.3) haplotypes. CONCLUSION: Several HLA-DRB1 and HLA-DQB1 alleles and haplotypes were proved to contribute to and protect from alloimmunization to Fya antigens. Alleles DRB1*04 and DRB1*15, as well as haplotypes DRB1*04-DQB1*03:02 and DRB1*15-DQB1*06:02 can be considered as risk factors, while allele DQB1*02 and haplotype DRB1*03-DQB1*02:01 have a protective role in Fya alloimmunization.

The origin of specificity and insight into recognition between an aminoacyl carrier protein and its partner ligase.

Acyl carrier proteins (ACPs) are among the most promiscuous proteins in terms of protein-protein interactions and it is quite puzzling how ACPs select the correct partner between many possible upstream and downstream binding proteins. To address this question, we performed molecular dynamics simulations on dimeric Bradyrhizobium japonicum Gly:CP ligase 1 to inspect the origin of its selectivity towards the three types of carrier proteins, namely holoCP, apoCP, and holoCP-Gly, which only differ in the attached prosthetic group. In line with experiments, MM-GBSA analysis revealed that the ligase preferentially binds the holoCP form to both subunits with the binding free energies of -20.7 and -19.1 kcal mol(-1), while the apoCP form, without the prosthetic group, is also recognized, but the binding values of -9.2 and -3.6 kcal mol(-1) suggest that there is no competition for the ligase binding as long as the holoCP is present. After the prosthetic group becomes glycylated, the holoCP-Gly dissociates from the ligase, as supported by its endergonic binding free energies of 2.9 and 20.9 kcal mol(-1). Our results indicate that these affinity differences are influenced by three aspects: the form of the prosthetic group and the specific non-polar hydrophobic interactions, as well as charge complementarity dominantly manifested through Arg220-Glu53 ion pair within the binding region among proteins. A careful examination of the bonding patterns within the ligase active site elucidated the interactions with Arg258, Asp215 and Tyr132 as being predominant in stabilizing the prosthetic group, which are significantly diminished upon glycation, thus promoting complex dissociation.

A single amino acid substitution affects the substrate specificity of the seryl-tRNA synthetase homologue.

Recently described and characterized Bradyrhizobium japonicum glycine:[carrier protein] ligase 1 (Bj Gly:CP ligase 1), a homologue of methanogenic type seryl-tRNA synthetase (SerRS) is an intriguing enzyme whose physiological role is not yet known. While aminoacyl-tRNA synthetases supply ribosome with amino acids for protein biosynthesis, this homologue transfers the activated amino acid to a specific carrier protein. Despite remarkable structural similarity between the Bj Gly:CP ligase 1 and the catalytic core domain of methanogenic type SerRS, the ligase displays altered and relaxed substrate specificity. In contrast to methanogenic SerRS which exclusively activates serine, the Bj Gly:CP ligase 1 predominantly activates glycine. Besides, it shows low activity in the presence of alanine, but it is incapable of activating serine. The detailed computational study aiming to address this unexpected substrate specificity toward the small aliphatic amino acids revealed the A281G Bj Gly:CP ligase 1 mutant as the most promising candidate with reconstituted catalytic activity toward the larger substrates. The A281G mutation is predicted to increase the active site volume, allowing alanine and serine to establish important hydrogen bonds within the active site, and to adopt an optimal orientation for the reaction. The results were tested by the site-directed mutagenesis experiments coupled with in vitro kinetic assays. It was found that the A281G substitution greatly affects the enzyme specificity and allows efficient activation of both polar and small aliphatic amino acids (serine, glycine and alanine), confirming predictions and conclusions based on molecular dynamics simulations.

Adaptation of aminoacyl-tRNA synthetase catalytic core to carrier protein aminoacylation.

Amino acid:[carrier protein] ligases (aa:CP ligases) are recently discovered enzymes that are highly similar to class II aminoacyl-tRNA synthetases (aaRSs). However, while aaRSs aminoacylate tRNA and supply building blocks for ribosomal translation, aa:CP ligases transfer activated amino acids to the phosphopantetheine group of small carrier proteins. We have solved the crystal structure of an aa:CP ligase complexed with the carrier protein (CP). The CP prosthetic group enters the active site from a different direction than tRNA in class II aaRS complexes through an idiosyncratic tunnel. CP binds to aa:CP ligase in a fundamentally different manner compared to tRNA binding by structurally closely related aaRSs. Based on crystallographic analysis, an enzyme of altered CP specificity was designed, and the mechanism of amino acid transfer to the prosthetic group was proposed. The presented study reveals how a conserved class II aaRS catalytic core can adapt to another function through minor structural alterations.

Substrate recognition and fidelity of maize seryl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the attachment of amino acids to their cognate tRNAs to establish the genetic code. To obtain the high degree of accuracy that is observed in translation, these enzymes must exhibit strict substrate specificity for their cognate amino acids and tRNAs. We studied the requirements for tRNA(Ser) recognition by maize cytosolic seryl-tRNA synthetase (SerRS). The enzyme efficiently recognized bacterial and eukaryotic tRNAs(Ser) indicating that it can accommodate various types of tRNA(Ser) structures. Discriminator base G73 is crucial for recognition by cytosolic SerRS. Although cytosolic SerRS efficiently recognized bacterial tRNAs(Ser), it is localized exclusively in the cytosol. The fidelity of maize cytosolic and dually targeted organellar SerRS with respect to amino acid recognition was compared. Organellar SerRS exhibited higher discrimination against tested non-cognate substrates as compared with cytosolic counterpart. Both enzymes showed pre-transfer editing activity implying their high overall accuracy. The contribution of various reaction pathways in the pre-transfer editing reactions by maize enzymes were different and dependent on the non-cognate substrate. The fidelity mechanisms of maize organellar SerRS, high discriminatory power and proofreading, indicate that aaRSs in general may play an important role in translational quality control in plant mitochondria and chloroplasts.

Homologs of aminoacyl-tRNA synthetases acylate carrier proteins and provide a link between ribosomal and nonribosomal peptide synthesis.

Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved enzymes catalyzing the formation of aminoacyl-tRNAs, that are used as substrates for ribosomal protein biosynthesis. In addition to full length aaRS genes, genomes of many organisms are sprinkled with truncated genes encoding single-domain aaRS-like proteins, which often have relinquished their canonical role in genetic code translation. We have identified the genes for putative seryl-tRNA synthetase homologs widespread in bacterial genomes and characterized three of them biochemically and structurally. The proteins encoded are homologous to the catalytic domain of highly diverged, atypical seryl-tRNA synthetases (aSerRSs) found only in methanogenic archaea and are deprived of the tRNA-binding domain. Remarkably, in comparison to SerRSs, aSerRS homologs display different and relaxed amino acid specificity. aSerRS homologs lack canonical tRNA aminoacylating activity and instead transfer activated amino acid to phosphopantetheine prosthetic group of putative carrier proteins, whose genes were identified in the genomic surroundings of aSerRS homologs. Detailed kinetic analysis confirmed that aSerRS homologs aminoacylate these carrier proteins efficiently and specifically. Accordingly, aSerRS homologs were renamed amino acid:[carrier protein] ligases (AMP forming). The enzymatic activity of aSerRS homologs is reminiscent of adenylation domains in nonribosomal peptide synthesis, and thus they represent an intriguing link between programmable ribosomal protein biosynthesis and template-independent nonribosomal peptide synthesis.

The proximal region of a noncatalytic eukaryotic seryl-tRNA synthetase extension is required for protein stability in vitro and in vivo.

Eukaryotic cytosolic seryl-tRNA synthetases (SerRS) have idiosyncratic C-terminal extensions not present in prokaryotic counterparts. The extensions of two eukaryotic SerRSs were subjected to mutagenesis and partial truncation. Only minor parts of the yeast or maize SerRS extensions, adjacent to the catalytic core (7 of 20 and 8 of 26 amino acids, respectively), were found to be indispensable for protein stability. Truncated proteins with substantially shortened extensions displayed unaltered catalytic properties and could complement a Saccharomyces cerevisiae strain with a disrupted SerRS gene, if these proximal regions were left intact. Although the yeast C-terminal SerRS extension is required for Pex21p binding, the maize counterpart with an appended yeast SerRS extension remained incapable of Pex21p binding, implying that additional regions of yeast SerRS may also contribute to the interaction with the peroxin. The proximal region of the eukaryotic SerRS C-terminal extension is indispensable for protein stability, while the remaining part of the extension remains available for other functions, such as species-specific protein:protein interactions.

Peroxin Pex21p interacts with the C-terminal noncatalytic domain of yeast seryl-tRNA synthetase and forms a specific ternary complex with tRNA(Ser).

The seryl-tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis-related factor Pex21p. Several deletion mutants of seryl-tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two-hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C-terminal amino acids abolished Pex21p binding to seryl-tRNA synthetase. The catalytic parameters of purified truncated seryl-tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C-terminally appended domain of yeast seryl-tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA-binding motif, but it instead participates in the formation of a specific ternary complex with seryl-tRNA synthetase and tRNA(Ser), strengthening the interaction of seryl-tRNA synthetase with its cognate tRNA(Ser).

Selective inhibition of divergent seryl-tRNA synthetases by serine analogues.

Seryl-tRNA synthetases (SerRSs) fall into two distinct evolutionary groups of enzymes, bacterial and methanogenic. These two types of SerRSs display only minimal sequence similarity, primarily within the class II conserved motifs, and possess distinct modes of tRNA(Ser) recognition. In order to determine whether the two types of SerRSs also differ in their recognition of the serine substrate, we compared the sensitivity of the representative methanogenic and bacterial-type SerRSs to serine hydroxamate and two previously unidentified inhibitors, serinamide and serine methyl ester. Our kinetic data showed selective inhibition of the methanogenic SerRS by serinamide, suggesting a lack of mechanistic uniformity in serine recognition between the evolutionarily distinct SerRSs.