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.

Bi-allelic mutations in HARS1 severely impair histidyl-tRNA synthetase expression and enzymatic activity causing a novel multisystem ataxic syndrome.

Mutations in histidyl-tRNA synthetase (HARS1), an enzyme that charges transfer RNA with the amino acid histidine in the cytoplasm, have only been associated to date with autosomal recessive Usher syndrome type III and autosomal dominant Charcot-Marie-Tooth disease type 2W. Using massive parallel sequencing, we identified bi-allelic HARS1 variants in a child (c.616G>T, p.Asp206Tyr and c.730delG, p.Val244Cysfs*6) and in two sisters (c.1393A>C, p.Ile465Leu and c.910_912dupTTG, p.Leu305dup), all characterized by a multisystem ataxic syndrome. All mutations are rare, segregate with the disease, and are predicted to have a significant effect on protein function. Functional studies helped to substantiate their disease-related roles. Indeed, yeast complementation assays showing that one out of two mutations in each patient is loss-of-function, and the reduction of messenger RNA and protein levels and enzymatic activity in patient's skin-derived fibroblasts, together support the pathogenicity of the identified HARS1 variants in the patient phenotypes. Thus, our efforts expand the allelic and clinical spectrum of HARS1-related disease.

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.

Peripheral neuropathy and cognitive impairment associated with a novel monoallelic HARS variant.

BACKGROUND: A 49-year-old male presented with late-onset demyelinating peripheral neuropathy, cerebellar atrophy, and cognitive deficit. Nerve biopsy revealed intra-axonal inclusions suggestive of polyglucosan bodies, raising the suspicion of adult polyglucosan bodies disease (OMIM 263570). METHODS AND RESULTS: While known genes associated with polyglucosan bodies storage were negative, whole-exome sequencing identified an unreported monoallelic variant, c.397G>T (p.Val133Phe), in the histidyl-tRNA synthetase (HARS) gene. While we did not identify mutations in genes known to be associated with polygucosan body disease, whole-exome sequencing revealed an unreported monoallelic variant, c.397G>T in the histidyl-tRNA synthetase (HARS) gene, encoding a substitution (Val133Phe) in the catalytic domain. Expression of this variant in patient cells resulted in reduced aminoacylation activity in extracts obtained from dermal fibroblasts, without compromising overall protein synthesis. INTERPRETATION: Genetic variants in the genes coding for the different aminoacyl-tRNA synthases are associated with various clinical conditions. To date, a number of HARS variant have been associated with peripheral neuropathy, but not cognitive deficits. Further studies are needed to explore why HARS mutations confer a neuronal-specific phenotype.

Knock-Down of Histidyl-tRNA Synthetase Causes Cell Cycle Arrest and Apoptosis of Neuronal Progenitor Cells in vivo.

Histidyl-tRNA Synthetase (HARS) is a member of the aminoacyl-tRNA synthetase family, which attach amino acids to their associated tRNA molecules. This reaction is a crucial step in protein synthesis that must be carried out in every cell of an organism. However, a number of tissue-specific, human genetic disorders have been associated with mutations in the genes for aminoacyl-tRNA synthetases, including HARS. These associations indicate that, while we know a great deal about the molecular and biochemical properties of this enzyme, we still do not fully understand how these proteins function in the context of an entire organism. To this end, we set out to knock-down HARS expression in the zebrafish and characterize the developmental consequences. Through our work we show that some tissues, particularly the nervous system, are more sensitive to HARS loss than others and we reveal a link between HARS and the proliferation and survival of neuronal progenitors during development.

Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish.

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.

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.

11th IUBMB Focused Meeting on the Aminoacyl-tRNA Synthetases: Sailing a New Sea of Complex Functions in Human Biology and Disease.

The 11th IUBMB Focused Meeting on Aminoacyl-tRNA Synthetases was held in Clearwater Beach, Florida from 29 October⁻2 November 2017, with the aim of presenting the latest research on these enzymes and promoting interchange among aminoacyl-tRNA synthetase (ARS) researchers. Topics covered in the meeting included many areas of investigation, including ARS evolution, mechanism, editing functions, biology in prokaryotic and eukaryotic cells and their organelles, their roles in human diseases, and their application to problems in emerging areas of synthetic biology. In this report, we provide a summary of the major themes of the meeting, citing contributions from the oral presentations in the meeting.

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.].

Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy.

Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.

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.