The catechol moiety of obafluorin is essential for antibacterial activity.

Obafluorin is a Pseudomonas fluorescens antibacterial natural product that inhibits threonyl-tRNA synthetase (ThrRS). It acts as a broad-spectrum antibiotic against a range of clinically relevant pathogens and comprises a strained ß-lactone ring decorated with catechol and 4-nitro-benzyl moieties. The catechol moiety is widespread in nature and its role in the coordination of ferric iron has been well-characterised in siderophores and Trojan horse antibiotics. Here we use a combination of mutasynthesis, bioassays, enzyme assays and metal binding studies to delineate the role of the catechol moiety in the bioactivity of obafluorin. We use P. fluorescens biosynthetic mutants to generate obafluorin analogues with modified catechol moieties. We demonstrate that an intact catechol is required for both antibacterial activity and inhibition of the ThrRS molecular target. Although recent work showed that the obafluorin catechol coordinates Zn2+ in the ThrRS active site, we find that obafluorin is a weak Zn2+ binder in vitro, contrasting with a strong, specific 1 : 1 interaction with Fe3+. We use bioassays with siderophore transporter mutants to probe the role of the obafluorin catechol in Fe3+-mediated uptake. Surprisingly, obafluorin does not behave as a Trojan horse antibiotic but instead exhibits increased antibacterial activity in the presence of Fe3+. We further demonstrate that Fe3+ binding prevents the hydrolytic breakdown of the ß-lactone ring, revealing a hitherto unreported function for the catechol moiety in natural product bioactivity.

Neuropathy-associated histidyl-tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish.

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.

De Novo and Bi-allelic Pathogenic Variants in NARS1 Cause Neurodevelopmental Delay Due to Toxic Gain-of-Function and Partial Loss-of-Function Effects.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.

Immunity-Guided Identification of Threonyl-tRNA Synthetase as the Molecular Target of Obafluorin, a ß-Lactone Antibiotic.

To meet the ever-growing demands of antibiotic discovery, new chemical matter and antibiotic targets are urgently needed. Many potent natural product antibiotics which were previously discarded can also provide lead molecules and drug targets. One such example is the structurally unique ß-lactone obafluorin, produced by Pseudomonas fluorescens ATCC 39502. Obafluorin is active against both Gram-positive and -negative pathogens; however, the biological target was unknown. We now report that obafluorin targets threonyl-tRNA synthetase, and we identify a homologue, ObaO, which confers immunity to the obafluorin producer. Disruption of obaO in P. fluorescens ATCC 39502 results in obafluorin sensitivity, whereas expression in sensitive E. coli strains confers resistance. Enzyme assays demonstrate that E. coli threonyl-tRNA synthetase is fully inhibited by obafluorin, whereas ObaO is only partly susceptible, exhibiting a very unusual partial inhibition mechanism. Altogether, our data highlight the utility of an immunity-guided approach for the identification of an antibiotic target de novo and will ultimately enable the generation of improved obafluorin variants.

Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics.

Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of new inhibitor frameworks, the use of semi-synthetic approaches combining chemistry and genome engineering, and more powerful techniques for identifying leads from the screening of large chemical libraries. Here, we review the inhibition of ARSs by small molecules, including the various families of natural products, as well as inhibitors developed by either rational design or high-throughput screening as antibiotics and anti-parasitic therapeutics.

Correction: A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

[This corrects the article DOI: 10.1371/journal.pone.0185317.].

A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

Histidyl tRNA Synthetase (HARS) is a member of the aminoacyl tRNA synthetase (ARS) family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.

The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNAHis binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry.

Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.

Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase.

The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsites within the catalytic domain of bacterial and human ThrRSs. These include the three substrate-binding sites for amino acid, ATP and tRNA associated with aminoacylation, and a fourth 'orthogonal' subsite created as a consequence of binding. Thus, borrelidin competes with all three aminoacylation substrates, providing a potent and redundant mechanism to inhibit ThrRS during protein synthesis. These results highlight a surprising natural design to achieve the quadrivalent inhibition of translation through a highly conserved family of enzymes.

Regulation of angiogenesis by aminoacyl-tRNA synthetases.

In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis.

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer.

BACKGROUND: Ovarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression. METHODS: Protein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α). RESULTS: There was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062). CONCLUSIONS: TARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.

Transfer RNA and human disease.

Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

Standardizing analysis of circulating microRNA: clinical and biological relevance.

Circulating microRNAs (c-miRNAs) provide a new dimension as clinical biomarkers for disease diagnosis, progression, and response to treatment. However, the discovery of individual miRNAs from biofluids that reliably reflect disease states is in its infancy. The highly variable nature of published studies exemplifies a need to standardize the analysis of miRNA in circulation. Here, we show that differential sample handling of serum leads to inconsistent and incomparable results. We present a standardized method of RNA isolation from serum that eliminates multiple freeze/thaw cycles, provides at least three normalization mechanisms, and can be utilized in studies that compare both archived and prospectively collected samples. It is anticipated that serum processed as described here can be profiled, either globally or on a gene by gene basis, for c-miRNAs and other non-coding RNA in the circulation to reveal novel, clinically relevant epigenetic signatures for a wide range of diseases.

Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis.

Aminoacyl-tRNA synthetases classically regulate protein synthesis but some also engage in alternative signaling functions related to immune responses and angiogenesis. Threonyl-tRNA synthetase (TARS) is an autoantigen in the autoimmune disorder myositis, and borrelidin, a potent inhibitor of TARS, inhibits angiogenesis. We explored a mechanistic link between these findings by testing whether TARS directly affects angiogenesis through inflammatory mediators. When human vascular endothelial cells were exposed to tumor necrosis factor-α (TNF-α) or vascular endothelial growth factor (VEGF), TARS was secreted into the cell media. Furthermore, exogenous TARS stimulated endothelial cell migration and angiogenesis in both in vitro and in vivo assays. The borrelidin derivative BC194 reduced the angiogenic effect of both VEGF and TARS, but not a borrelidin-resistant TARS mutant. Our findings reveal a previously undiscovered function for TARS as an angiogenic, pro-migratory extracellular signaling molecule. TARS thus provides a potential target for detecting or interdicting disease-related inflammatory or angiogenic responses.

The α-amino group of the threonine substrate as the general base during tRNA aminoacylation: a new version of substrate-assisted catalysis predicted by hybrid DFT.

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.

Fidelity escape by the unnatural amino acid ß-hydroxynorvaline: an efficient substrate for Escherichia coli threonyl-tRNA synthetase with toxic effects on growth.

In all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-tRNA synthetases (ARSs). Some nonproteogenic amino acids, including ß-hydroxynorvaline (HNV) are nevertheless efficiently aminoacylated and become incorporated into proteins. To investigate the basis of HNV's ability to function in protein synthesis, the utilization of HNV by Escherichia coli threonyl-tRNA synthetase (ThrRS) was investigated through both in vitro functional experiments and bacterial growth studies. The measured specificity constant (k(cat)/K(M)) for HNV was found to be only 20-30-fold less than that of cognate threonine. The rate of aminoacyl transfer (10.4 s(-1)) was 10-fold higher than the multiple turnover k(cat) value (1 s(-1)), indicating that, as for cognate threonine, amino acid activation is likely to be the rate-limiting step. Like noncognate serine, HNV enhances the ATPase function of the synthetic site, at a rate not increased by nonaminoacylatable (3'-dA76) tRNA. ThrRS also failed to exhibit posttransfer editing activity against HNV. In growing bacteria, the addition of HNV dramatically suppressed growth rates, which indicates either negative phenotypic consequences associated with its incorporation into protein or inhibition of an unidentified metabolic reaction. The inability of wild ThrRS to prevent utilization of HNV as a substrate illustrates that, for at least one ARS, the naturally occurring enzyme lacks the capability to effectively discriminate against nonproteogenic amino acids that are not encountered under normal physiological conditions. Other examples of "fidelity escape" in the ARSs may serve as useful starting points in the design of ARSs with specificity for unnatural amino acids.