MACSPI enables tissue-selective proteomic and interactomic analyses in multicellular organisms.

Multicellular organisms are composed of many tissue types that have distinct morphologies and functions, which are largely driven by specialized proteomes and interactomes. To define the proteome and interactome of a specific type of tissue in an intact animal, we developed a localized proteomics approach called Methionine Analog-based Cell-Specific Proteomics and Interactomics (MACSPI). This method uses the tissue-specific expression of an engineered methionyl-tRNA synthetase to label proteins with a bifunctional amino acid 2-amino-5-diazirinylnonynoic acid in selected cells. We applied MACSPI in Caenorhabditis elegans, a model multicellular organism, to selectively label, capture, and profile the proteomes of the body wall muscle and the nervous system, which led to the identification of tissue-specific proteins. Using the photo-cross-linker, we successfully profiled HSP90 interactors in muscles and neurons and identified tissue-specific interactors and stress-related interactors. Our study demonstrates that MACSPI can be used to profile tissue-specific proteomes and interactomes in intact multicellular organisms.

Bacterial Hydrazine Biosynthetic Pathways Featuring Cupin/Methionyl tRNA Synthetase-like Enzymes.

Nitrogen-Nitrogen (N-N) bond-containing functional groups in natural products and synthetic drugs play significant roles in exerting biological activities. The mechanisms of N-N bond formation in natural organic molecules have garnered increasing attention over the decades. Recent advances have illuminated various enzymatic and nonenzymatic strategies, and our understanding of natural N-N bond construction is rapidly expanding. A group of didomain proteins with zinc-binding cupin/methionyl-tRNA synthetase (MetRS)-like domains, also known as hydrazine synthetases, generates amino acid-based hydrazines, which serve as key biosynthetic precursors of diverse N-N bond-containing functionalities such as hydrazone, diazo, triazene, pyrazole, and pyridazinone groups. In this review, we summarize the current knowledge on hydrazine synthetase mechanisms and the various pathways employing this unique bond-forming machinery.

Phylogeny-guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase-like Enzymes.

Cupin/methionyl-tRNA synthetase (MetRS)-like didomain enzymes catalyze nitrogen-nitrogen (N-N) bond formation between Nω-hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N-N bonds. While the combination of these two building blocks leads to the creation of diverse hydrazine products, the full extent of their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny-guided genome-mining of related hydrazine biosynthetic pathways consisting of two enzymes: flavin-dependent Nω-hydroxylating monooxygenases (NMOs) that produce Nω-hydroxylamine precursors and cupin/MetRS-like enzymes that couple the Nω-hydroxylamines with amino acids via N-N bonds. A phylogenetic analysis identified the largely unexplored sequence spaces of these enzyme families. The biochemical characterization of NMOs demonstrated their capabilities to produce various Nω-hydroxylamines, including those previously not known as precursors of N-N bonds. Furthermore, the characterization of cupin/MetRS-like enzymes identified five new hydrazine products with novel combinations of building blocks, including one containing non-amino acid building blocks: 1,3-diaminopropane and putrescine. This study substantially expanded the variety of N-N bond forming pathways mediated by cupin/MetRS-like enzymes.

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein.

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)-like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip-ERS-QRS) and the M-complex (tRip-ERS-MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host-pathogen interaction is discussed.

Monitoring regional astrocyte diversity by cell type-specific proteomic labeling in vivo.

Astrocytes exhibit regional heterogeneity in morphology, function and molecular composition to support and modulate neuronal function and signaling in a region-specific manner. To characterize regional heterogeneity of astrocytic proteomes of different brain regions we established an inducible Aldh1l1-methionyl-tRNA-synthetaseL274G (MetRSL274G ) mouse line that allows astrocyte-specific metabolic labeling of newly synthesized proteins by azidonorleucine (ANL) in vivo and subsequent isolation of tagged proteins by click chemistry. We analyzed astrocytic proteins from four different brain regions by mass spectrometry. The induced expression of MetRSL274G is restricted to astrocytes and identified proteins show a high overlap with proteins compiled in "AstroProt," a newly established database for astrocytic proteins. Gene enrichment analysis reveals a high similarity among brain regions with subtle differences in enriched biological processes and in abundances of key astrocytic proteins for hippocampus, cortex and striatum. However, the cerebellar proteome stands out with proteins being highly associated with the calcium signaling pathway or with bipolar disorder. Subregional analysis of single astrocyte TAMRA intensities in hippocampal layers indicates distinct subregional heterogeneity of astrocytes and highlights the applicability of our toolbox to study differences of astrocytic proteomes in vivo.

Protein instability associated with AARS1 and MARS1 mutations causes trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable.

Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase.

Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 'writes' modifications on tRNA and mRNA, and both types of RNAs are 'read' by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.

Molecular basis for diaryldiamine selectivity and competition with tRNA in a type 2 methionyl-tRNA synthetase from a Gram-negative bacterium.

Gram-negative bacteria are responsible for a variety of human, animal, and plant diseases. The spread of multidrug-resistant Gram-negative bacteria poses a challenge to disease control and highlights the need for novel antimicrobials. Owing to their critical role in protein synthesis, aminoacyl-tRNA synthetases, including the methionyl-tRNA synthetases MetRS1 and MetRS2, are attractive drug targets. MetRS1 has long been exploited as a drug target in Gram-positive bacteria and protozoan parasites. However, MetRS1 inhibitors have limited action upon Gram-negative pathogens or on Gram-positive bacteria that produce MetRS2 enzymes. The underlying mechanism by which MetRS2 enzymes are insensitive to MetRS1 inhibitors is presently unknown. Herein, we report the first structures of MetRS2 from a multidrug-resistant Gram-negative bacterium in its ligand-free state and bound to its substrate or MetRS1 inhibitors. The structures reveal the binding mode of two diaryldiamine MetRS1 inhibitors that occupy the amino acid-binding site and a surrounding auxiliary pocket implicated in tRNA acceptor arm binding. The structural features associated with amino acid polymorphisms found in the methionine and auxiliary pockets reveal the molecular basis for diaryldiamine binding and selectivity between MetRS1 and MetRS2 enzymes. Moreover, we show that mutations in key polymorphic residues in the methionine and auxiliary pockets not only altered inhibitor binding affinity but also significantly reduced enzyme function. Our findings thus reinforce the tRNA acceptor arm binding site as a druggable pocket in class I aminoacyl-tRNA synthetases and provide a structural basis for optimization of MetRS2 inhibitors for the development of new antimicrobials against Gram-negative pathogens.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

Increased Mars2 expression upon microRNA-4661-5p-mediated KDM5D downregulation is correlated with malignant degree of gastric cancer cells.

Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related mortality worldwide. Methionyl-tRNA synthetase 2 (Mars2) has been suggested as a biomarker indicating poor prognosis of cancers. This study focuses on the function of Mars2 in GC and the responsible molecules. Mars2 was highly expressed in GC patients according to a transcriptome analysis and the data from the public database, and its high expression was confirmed in the acquired GC cell lines. Downregulation of Mars2 significantly weakened the proliferation, resistance to death, migration and invasion of GC cells. The H3K4me3 modification level was increased in the promoter region of Mars2, which was attributed to reduced abundance of lysine demethylase 5D (KDM5D) in the Mars2 promoter. MicroRNA (miR)-4661-5p was identified as an upstream regulator of KDM5D. Downregulation of miR-4661-5p led to an increase in the expression of KDM5D while a decline in the expression of Mars2, which reduced the malignant behaviors of GC cells; however, the malignant behaviors of GC cells was restored after further inhibition of KDM5D. To conclude, this study suggested that increased Mars2 expression upon miR-4661-5p-mediated KDM5D downregulation is correlated with malignant degree of GC cells.

Temperature dependent mistranslation in a hyperthermophile adapts proteins to lower temperatures.

All organisms universally encode, synthesize and utilize proteins that function optimally within a subset of growth conditions. While healthy cells are thought to maintain high translational fidelity within their natural habitats, natural environments can easily fluctuate outside the optimal functional range of genetically encoded proteins. The hyperthermophilic archaeon Aeropyrum pernix (A. pernix) can grow throughout temperature variations ranging from 70 to 100°C, although the specific factors facilitating such adaptability are unknown. Here, we show that A. pernix undergoes constitutive leucine to methionine mistranslation at low growth temperatures. Low-temperature mistranslation is facilitated by the misacylation of tRNA(Leu) with methionine by the methionyl-tRNA synthetase (MetRS). At low growth temperatures, the A. pernix MetRS undergoes a temperature dependent shift in tRNA charging fidelity, allowing the enzyme to conditionally charge tRNA(Leu) with methionine. We demonstrate enhanced low-temperature activity for A. pernix citrate synthase that is synthesized during leucine to methionine mistranslation at low-temperature growth compared to its high-fidelity counterpart synthesized at high-temperature. Our results show that conditional leucine to methionine mistranslation can make protein adjustments capable of improving the low-temperature activity of hyperthermophilic proteins, likely by facilitating the increasing flexibility required for greater protein function at lower physiological temperatures.

Global tRNA misacylation induced by anaerobiosis and antibiotic exposure broadly increases stress resistance in Escherichia coli.

High translational fidelity is commonly considered a requirement for optimal cellular health and protein function. However, recent findings have shown that inducible mistranslation specifically with methionine engendered at the tRNA charging level occurs in mammalian cells, yeast and archaea, yet it was unknown whether bacteria were capable of mounting a similar response. Here, we demonstrate that Escherichia coli misacylates non-methionyl-tRNAs with methionine in response to anaerobiosis and antibiotic exposure via the methionyl-tRNA synthetase (MetRS). Two MetRS succinyl-lysine modifications independently confer high tRNA charging fidelity to the otherwise promiscuous, unmodified enzyme. Strains incapable of tRNA mismethionylation are less adept at growth in the presence of antibiotics and stressors. The presence of tRNA mismethionylation and its potential role in mistranslation within the bacterial domain establishes this response as a pervasive biological mechanism and connects it to diverse cellular functions and modes of fitness.

Modulating the Structure and Function of an Aminoacyl-tRNA Synthetase Cofactor by Biotinylation.

Arc1p is a yeast-specific tRNA-binding protein that forms a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the cytoplasm to regulate their catalytic activities and subcellular distributions. Despite Arc1p not being involved in any known biotin-dependent reaction, it is a natural target of biotin modification. Results presented herein show that biotin modification had no obvious effect on the growth-supporting activity, subcellular distribution, tRNA binding, or interactions of Arc1p with GluRSc and MetRS. Nevertheless, biotinylation of Arc1p was temperature dependent; raising the growth temperature from 30 to 37 °C drastically reduced its biotinylation level. As a result, Arc1p purified from a yeast culture that had been grown overnight at 37 °C was essentially biotin free. Non-biotinylated Arc1p was more heat stable, more flexible in structure, and more effective than its biotinylated counterpart in promoting glutamylation activity of the otherwise inactive GluRSc at 37 °C in vitro Our study suggests that the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes.

Binding Properties of Split tRNA to the C-terminal Domain of Methionyl-tRNA Synthetase of Nanoarchaeum equitans.

The C-terminal domain of methionyl-tRNA synthetase (MetRS-C) from Nanoarchaeum equitans is homologous to a tRNA-binding protein consisting of 111 amino acids (Trbp111) from Aquifex aeolicus. The crystal structure of MetRS-C showed that it existed as a homodimer, and that each monomer possessed an oligonucleotide/oligosaccharide-binding fold (OB-fold). Analysis using a quartz crystal microbalance indicated that MetRS-C freshly isolated from N. equitans was bound to tRNA. However, binding of the split 3'-half tRNA species was stronger than that of the 5'-half species. The T-loop and the 3'-end regions of the split 3'-half tRNA were found to be responsible for the binding. The minimum structure for binding to MetRS-C might be a minihelix-like stem-loop with single-stranded 3'-terminus. After successive duplications of such a small hairpin structure with the assistance of a Trbp-like structure, the interaction of the T-loop region of the 3'-half with a Trbp-like structure could have been evolutionarily replaced by RNA-RNA interactions, along with many combinational tertiary interactions, to form the modern tRNA structure.

tRNA Misacylation with Methionine in the Mouse Gut Microbiome in Situ.

Global protein mistranslation with methionine has been shown to be a conserved biological process that affords distinct functional advantages in all three domains of life. In all instances, methionine mistranslation occurs through a regulated process where low-fidelity forms of methionyl-tRNA synthetase are conditionally induced to mischarge non-methionyl-tRNAs with methionine followed by the utilization of the misacylated tRNAs in translation. In mammals, methionine mistranslation contributes to oxidative stress response; in the hyperthermophilic archaeon Aeropyrum pernix, methionine mistranslation produces proteins that are better adapted to low temperature growth; in E. coli, methionine mistranslation increases resistance to antibiotics and chemical stressors. The phenotypic benefits conferred by tRNA mismethionylation suggest that it should be a widespread adaptational mechanism in diverse bacterial lineages, yet this response has only been described in E. coli. Furthermore, previous microbial investigations on this response have been confined to axenic laboratory cultures. It was unknown whether tRNA mismethionylation was relevant in a natural microbial habitat. Here we show that four abundant gut microbiotal genera belonging to the Firmicutes and Bacteroidetes phyla perform constitutive tRNA misacylation with methionine in the mouse cecum in situ. These results reveal the ubiquity of the tRNA mismethionylation process among bacteria and implicate the potential importance of this response for subsistence and adaptation in natural habitats.

Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC50 of 4nM.

Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis.

Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNA(Met) in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.

Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNA(Met), leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

Intriguing cellular processing of a fluorinated amino acid during protein biosynthesis in Escherichia coli.

Bioincorporation of the methionine analogue S-(2-fluoroethyl)-l-homocysteine (l-MFE) into bacteriophage lysozyme overproduced in Escherichia coli results not only in the expected l-MFE incorporation but surprisingly substantial l-vinthionine incorporation into the labeled lysozymes. Synthetic l-vinthionine itself however is not activated by purified Escherichia coli methionyl-tRNA synthetase. The indirect preparation of vinthionine-containing proteins has the potential to be an alternate strategy to prepare vinyl thioether moieties for click chemistry applications on proteins.

A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.