Bioorthogonal non-canonical amino acid tagging to track transplanted human induced pluripotent stem cell-specific proteome.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo. METHODS: In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts. RESULTS: We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs. CONCLUSION: The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

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.

Successful lung transplantation in genetic methionyl-tRNA synthetase-related alveolar proteinosis/lung fibrosis without recurrence under methionine supplementation: Medium-term outcome in 4 cases.

Pulmonary alveolar proteinosis (PAP) results from the accumulation of lipoproteinaceous material in the alveoli and alveolar macrophages, and can be associated with pulmonary fibrosis, with a need for lung transplantation (LTx). Causes of PAP are autoimmune (90%-95%), secondary (5%), or hereditary (<1%). Patients with hereditary PAP are generally not considered for isolated LTx, due to the high probability of recurrence after LTx, and only a challenging scenario with sequential LTx followed by hematopoietic stem cell transplantation (HSCT) was reported as successful. Recently, a new genetic cause of PAP linked to mutations in the methionyl-tRNA synthetase (MARS) gene has been reported, with a highly variable clinical presentation. Because clinical correction of the defective MARS activity with methionine supplementation has been reported in nontransplanted children, we reassessed the feasibility of LTx for candidates with MARS-related PAP/fibrosis. We report 3 cases of LTx performed for MARS-related pulmonary alveolar proteinosis-pulmonary fibrosis without recurrence under methionine supplementation, whereas another fourth case transplanted without supplementation had fatal PAP recurrence. These results suggest the effectiveness of methionine in correcting defective MARS activity and also looking for this very rare diagnosis in case of unclassified PAP/fibrosis. It argues for not excluding the feasibility of isolated LTx in patients with MARS mutation.

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.

Biochemical and structural characterization of chlorhexidine as an ATP-assisted inhibitor against type 1 methionyl-tRNA synthetase from Gram-positive bacteria.

Methionyl-tRNA synthetase (MetRS) catalyzes the attachment of l-methionine (l-Met) to tRNAMet to generate methionyl-tRNAMet, an essential substrate for protein translation within ribosome. Owing to its indispensable biological function and the structural discrepancies with human counterpart, bacterial MetRS is considered an ideal target for developing antibacterials. Herein, chlorhexidine (CHX) was identified as a potent binder of Staphylococcus aureus MetRS (SaMetRS) through an ATP-aided affinity screening. The co-crystal structure showed that CHX simultaneously occupies the enlarged l-Met pocket (EMP) and the auxiliary pocket (AP) of SaMetRS with its two chlorophenyl groups, while its central hexyl linker swings upwards to interact with some conserved hydrophobic residues. ATP adopts alternative conformations in the active site cavity, and forms ionic bonds and water-mediated hydrogen bonds with CHX. Consistent with this synergistic binding mode, ATP concentration-dependently enhanced the binding affinity of CHX to SaMetRS from 10.2 µM (no ATP) to 0.45 µM (1 mM ATP). While it selectively inhibited two representative type 1 MetRSs from S. aureus and Enterococcus faecalis, CHX did not show significant interactions with three tested type 2 MetRSs, including human cytoplasmic MetRS, in the enzyme inhibition and biophysical binding assays, probably due to the conformational differences between two types of MetRSs at their EMP and AP. Our findings on CHX may inspire the design of MetRS-directed antimicrobials in future.

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.

Proteomic analysis of meropenem-induced outer membrane vesicles released by carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important multidrug resistance (MDR) pathogen that threatens human health and is the main source of hospital-acquired infection. Outer membrane vesicles (OMVs) are extracellular vesicles derived from Gram-negative bacteria and contain materials involved in bacterial survival and pathogenesis. They also contribute to cellular communication to nearby or distant recipient cells and influence their functions and phenotypes. In this study, we sought to understand the mechanism of bacterial response to meropenem pressure and explore the relationship between pathogenic proteins and the high pathogenicity of bacteria. We performed whole-genome PacBio sequencing on a clinical CRKP strain, and its OMVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and proteomic analysis. Thousands of vesicle proteins have been identified in mass spectrometry-based high-throughput proteomics analyses of K. pneumoniae OMVs. Protein functionality analysis showed that the OMVs were predominantly involved in metabolic, intracellular compartments, nucleic acid binding, survival, defense, and antibiotic resistance, such as Chromosome partition protein MukB, 3-methyl-2-oxobutanoate hydroxymethyltransferase, methionine-tRNA ligase, Heat shock protein 60 family chaperone GroEL, and Gamma-glutamyl phosphate reductase. Additionally, a protein-protein interaction network demonstrated that OMVs from meropenem-treated K. pneumoniae showed the highest connectivity in DNA polymerase I, phenylalanine-tRNA ligase beta subunit, DNA-directed RNA polymerase subunit beta, methionine-tRNA ligase, DNA-directed RNA polymerase subunit beta, and DNA-directed RNA polymerase subunit alpha. The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-treated K. pneumoniae, which provides clues for revealing the biogenesis and pathophysiological functions of Gram-negative bacteria OMVs. The significant differentially expressed proteins identified in this study are of great significance for exploring effective control strategies for CRKP infection.IMPORTANCEMeropenem is one of the main antibiotics used in the clinical treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP). This study demonstrated that some important metabolic changes occurred in meropenem-induced CRKP-outer membrane vesicles (OMVs), The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-induced Klebsiella pneumoniae. Furthermore, this is the first study to discuss the protein-protein interaction network of the OMVs released by CRKP, especially under antibiotic stress.

Crystal structure of Methionyl-tRNA Synthetase from Rickettsia typhi in complex with its cognate amino acid.

Rickettsia typhi is the causative agent of murine typhus (endemic typhus), a febrile illness that can be self-contained, though in some cases it can progress to death. The three dimensional structure of Methionyl-tRNA Synthetase from R. typhi (RtMetRS) in complex with its substrate l-methionine was solved by molecular replacement and refined at 2.30 Å resolution in space group P1 from one X-ray diffraction dataset. Processing and refinement trials were decisive to establish the lower symmetry space group and indicated the presence of twinning with four domains. RtMetRS belongs to the MetRS1 family and was crystallized with the CP domain in an open conformation, what is distinctive from other MetRS1 enzymes whose structures were solved with a bound L-methionine (therefore, in a closed conformation). This conformation resembles the ones observed in the MetRS2 family.

A mutant methionyl-tRNA synthetase-based toolkit to assess induced-mesenchymal stromal cell secretome in mixed-culture disease models.

BACKGROUND: Mesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRSL274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation. METHODS: We used CRISPR/Cas9 homology-directed repair to stably integrate MetRSL274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRSL274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRSL274G-expressing iMSCs with naïve or lipopolysaccharide (LPS)-treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays. RESULTS: Our results showed successful integration of MetRSL274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRSL274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells. CONCLUSIONS: The MetRSL274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.

Redesigning methionyl-tRNA synthetase for ß-methionine activity with adaptive landscape flattening and experiments.

Amino acids (AAs) with a noncanonical backbone would be a valuable tool for protein engineering, enabling new structural motifs and building blocks. To incorporate them into an expanded genetic code, the first, key step is to obtain an appropriate aminoacyl-tRNA synthetase. Currently, directed evolution is not available to optimize AAs with noncanonical backbones, since an appropriate selective pressure has not been discovered. Computational protein design (CPD) is an alternative. We used a new CPD method to redesign MetRS and increase its activity towards ß-Met, which has an extra backbone methylene. The new method considered a few active site positions for design and used a Monte Carlo exploration of the corresponding sequence space. During the exploration, a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to their ß-Met binding affinities. Eighteen predicted variants were chosen for experimental testing; 10 exhibited detectable activity for ß-Met adenylation. Top predicted hits were characterized experimentally in detail. Dissociation constants, catalytic rates, and Michaelis constants for both α-Met and ß-Met were measured. The best mutant retained a preference for α-Met over ß-Met; however, the preference was reduced, compared to the wildtype, by a factor of 29. For this mutant, high resolution crystal structures were obtained in complex with both α-Met and ß-Met, indicating that the predicted, active conformation of ß-Met in the active site was retained.

Computational Insight into Substrate-Induced Conformational Changes in Methionyl-tRNA Synthetase of Mycobacterium Tuberculosis.

Tuberculosis caused by Mycobacterium tuberculosis (M.tb) has killed millions worldwide. Antibiotic resistance leads to the ineffectiveness of the current therapies. Aminoacyl tRNA synthetase (aaRS) class of proteins involved in protein synthesis are promising bacterial targets for developing new therapies. Here, we carried out a systematic comparative study on the aaRS sequences from M.tb and human. We listed important M.tb aaRS that could be explored as potential M.tb targets alongside the detailed conformational space analysis of methionyl-tRNA synthetase (MetRS) in apo- and substrate-bound form, which is among the proposed targets. Understanding the conformational dynamics is central to the mechanistic understanding of MetRS, as the substrate binding leads to the conformational changes causing the reaction to proceed. We performed the most complete simulation study of M.tb MetRS for 6 microseconds (2 systems × 3 runs × 1 microsecond) in the apo and substrate-bound states. Interestingly, we observed differential features, showing comparatively large dynamics for the holo simulations, whereas the apo structures became slightly compact with reduced solvent exposed area. In contrast, the ligand size decreased significantly in holo structures possibly to relax ligand conformation. Our findings correlate with experimental studies, thus validating our protocol. Adenosine monophosphate moiety of the substrate exhibited quite higher fluctuations than the methionine. His21 and Lys54 were found to be the important residues forming prominent hydrogen bond and salt-bridge interactions with the ligand. The ligand-protein affinity decreased during simulations as computed by MMGBSA analysis over the last 500 ns trajectories, which indicates the conformational changes upon ligand binding. These differential features could be further explored for designing new M.tb inhibitors.

MARS2 drives metabolic switch of non-small-cell lung cancer cells via interaction with MCU.

Mitochondrial methionyl-tRNA synthetase (MARS2) canonically mediates the formation of fMet-tRNAifMet for mitochondrial translation initiation. Mitochondrial calcium uniporter (MCU) is a major gate of Ca2+ flux from cytosol into the mitochondrial matrix. We found that MARS2 interacts with MCU and stimulates mitochondrial Ca2+ influx. Methionine binding to MARS2 would act as a molecular switch that regulates MARS2-MCU interaction. Endogenous knockdown of MARS2 attenuates mitochondrial Ca2+ influx and induces p53 upregulation through the Ca2+-dependent CaMKII/CREB signaling. Subsequently, metabolic rewiring from glycolysis into pentose phosphate pathway is triggered and cellular reactive oxygen species level decreases. This metabolic switch induces inhibition of epithelial-mesenchymal transition (EMT) via cellular redox regulation. Expression of MARS2 is regulated by ZEB1 transcription factor in response to Wnt signaling. Our results suggest the mechanisms of mitochondrial Ca2+ uptake and metabolic control of cancer that are exerted by the key factors of the mitochondrial translational machinery and Ca2+ homeostasis.

MARS1 mutations linked to familial trigeminal neuralgia via the integrated stress response.

BACKGROUND: While new genetic analysis methods are widely used in the clinic, few researchers have focused on trigeminal neuralgia (TN) with familial clustering (≥ 2 TN patients in one kindred family). Previous literature suggests that familial trigeminal neuralgia (FTN) may be associated with inherited genetic factors. To date, few next-generation sequencing studies have been reported for FTN. This study investigated the pathogenic mechanism of FTN by using whole-exome sequencing (WES) technology, which may enhance our understanding of human TN pathophysiology.  METHOD: We performed WES for 7 probands from families of FTN. Sanger sequencing was performed for two control groups (FTN family members group and nonfamilial TN subject group) to potentially identify new FTN-related gene mutations. In families where FTN probands carried potentially pathogenic gene mutations, the ribonucleic acid (RNA) of FTN probands and related family members, as well as nonfamilial TN patients were analysed by RNA sequencing (RNA-seq) to confirm differential gene expression. RESULTS: Seven probands were derived from 3 Chinese families. WES and Sanger sequencing identified MARS1 mutation c.2398C > A p.(Pro800Thr) in Family 1. MARS1 mutation was confirmed in 14/26 [53.8%] members of Family 1 in FTN family member group, while none of nonfamilial TN subjects had this MARS1 mutation. RNA-seq showed that 3 probands in Family 1 had higher expression of Fosl1 (Fos-like antigen 1) and NFE2 (Nuclear factor, erythroid 2) than 3 subjects in the nonfamilial TN subject group. Fosl1 and NFE2 are genes related to integrated stress response (ISR). CONCLUSION: MARS1 mutations may cause chronic activation of ISR, contribute to ISR pathophysiological changes in FTN, and cause/accelerate peripheral nerve degeneration. The findings of this study can enrich our knowledge of the role of molecular genetics in TN in humans.

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.

Probing interactions of aminoacyl-adenylate with Mycobacterium tuberculosis methionyl-tRNA synthetase through in silico site-directed mutagenesis and free energy calculation.

Methionyl-tRNA synthetase (MetRS) is an attractive molecular target for antibiotic discovery. Recently, we have developed several classes of small-molecular inhibitors of Mycobacterium tuberculosis MetRS possessing antibacterial activity. In this article, we performed in silico site-directed mutagenesis of aminoacyl-adenylate binding site of M. tuberculosis MetRS in order to identify crucial amino acid residues for substrate interaction. The umbrella sampling algorithm was used to calculate the binding free energy (ΔG) of these mutated forms with methionyl-adenylate analogue. According to the obtained results, the replacement of Glu24 and Leu293 by alanine leads to the most significant decrease in the binding free energy (ΔG) for adenylate analogue with methionyl-tRNA synthetase indicating increasing of the affinity, which in turn causes the loss of compounds inhibitory activity. Therefore, these amino acid residues can be proposed for further experimental site-directed mutagenesis to confirm binding mode of inhibitors and should be taken into account during chemical optimization to overcome resistance due to mutations.Communicated by Ramaswamy H. Sarma.

In-silico, synthesis, structure elucidation and anticancer activity study of 2-(3,4-dichlorophenyl)-4H-benzo[d][1,3]oxazin-4-one.

The research aims to synthesize 2-(3,4-dichlorophenyl)-4H-benzo[d][1,3]oxazin-4-one and evaluate its anticancer activity against MCF-7. This compound was selected based on in-silico study conducted against several dihalophenylbenzoxazinone analogues using molecular docking towards Methionyl-tRNA synthetase. Synthesis of target compound was carried out using anthranilic acid and 3,4-dichlorobenzoyl chloride. The resulting compound was characterized using various spectroscopic analysis: 1D and 2D NMR, infrared and MS. In-silico studies was performed by MVD. Several designed compounds were docked into the active site on Methionyl-tRNA Synthetase (1PG2). Anticancer activity was evaluated by MTT Assay against MCF-7. 2-(3,4-dichlorophenyl)-4H-benzo[d][1,3]oxazin-4-one has been successfully synthesized with decent amount of yield 88%. Its spectroscopic analysis 1D and 2D NMR, MS, FTIR has proven the chemical structure of compound. In-silico studies toward the enzyme showed docking score of -76.04 Kcal/mol, higher than its native ligand (-93.50 Kcal/mol). Meanwhile, MTT assay result against MCF-7 showed IC50 value of 68.59ppm. Based on preliminary in-silico studies inhibited Methionyl-tRNA Synthetase, 2-(3,4-dichlorophenyl)-4H-benzo[d][1,3]oxazin-4-one was synthesized and tested in-vitro against MCF-7. Albeit the compound does not possess better docking score than native ligand, it is still argued that benzoxazine ring can be considered as a potential anticancer agent, as showed by MTT assay result which indicated moderate cytotoxicity.

Molecular Modeling, Virtual Screening, and Molecular Dynamics for Leishmania infantum Methionyl-tRNA Synthetase.

Visceral leishmaniasis is a neglected tropical disease (NTD) caused by Leishmania infantum and L. donovani that is lethal in cases of nontreatment. The treatments are limited by serious drawbacks involving safety, resistance, stability, and high costs. In this work, we aimed to identify inhibitors of Leishmania infantum methionyl-tRNA synthetase (LiMetRS), a validated molecular target for leishmaniasis drug discovery, using a combination of strategies. A virtual database of compounds was organized by filtering compounds from the ZINC15 database. Homology modeling was used to obtain the structure of LiMetRS based on the crystal coordinates of the enzyme from Trypanosoma brucei (TbMetRS). A virtual screening using molecular docking identified 10 candidate compounds from among more than 5 million that were included in the initial database. The selected hits were further evaluated using a script created in this work to select only the ligands that interacted with specific amino acids in the catalytic site of the enzyme. Furthermore, suitable pharmacokinetic profiles were predicted for the selected compounds, especially a good balance between aqueous solubility and lipophilic character, no ability to cross the blood-brain barrier, good oral absorption, and no liability toward P-gp efflux for most compounds. Six compounds were then subjected to all-atom molecular dynamics. Two compounds showed good stability when bound to the leishmanial enzyme, which provided a deeper understanding of the structural differences between TbMetRS and LiMetRS that can guide further drug discovery efforts for visceral leishmaniasis.

Characterization of the Secretome of a Specific Cell Expressing Mutant Methionyl-tRNA Synthetase in Co-Culture Using Click Chemistry.

Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.

Fragment screening and structural analyses highlight the ATP-assisted ligand binding for inhibitor discovery against type 1 methionyl-tRNA synthetase.

Methionyl-tRNA synthetase (MetRS) charges tRNAMet with l-methionine (L-Met) to decode the ATG codon for protein translation, making it indispensable for all cellular lives. Many gram-positive bacteria use a type 1 MetRS (MetRS1), which is considered a promising antimicrobial drug target due to its low sequence identity with human cytosolic MetRS (HcMetRS, which belongs to MetRS2). Here, we report crystal structures of a representative MetRS1 from Staphylococcus aureus (SaMetRS) in its apo and substrate-binding forms. The connecting peptide (CP) domain of SaMetRS differs from HcMetRS in structural organization and dynamic movement. We screened 1049 chemical fragments against SaMetRS preincubated with or without substrate ATP, and ten hits were identified. Four cocrystal structures revealed that the fragments bound to either the L-Met binding site or an auxiliary pocket near the tRNA CCA end binding site of SaMetRS. Interestingly, fragment binding was enhanced by ATP in most cases, suggesting a potential ATP-assisted ligand binding mechanism in MetRS1. Moreover, co-binding with ATP was also observed in our cocrystal structure of SaMetRS with a class of newly reported inhibitors that simultaneously occupied the auxiliary pocket, tRNA site and L-Met site. Our findings will inspire the development of new MetRS1 inhibitors for fighting microbial infections.

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.