tyrosyl-tRNA Synthetase Research Articles

A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic.

The Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli-based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine.

LncRNA NCK1-AS1 Aggravates Hepatocellular Carcinoma by the miR-22-3p/YARS Axis to Activate PI3K/AKT Signaling.

Hepatocellular carcinoma (HCC) is frequently diagnosed at late stages when curative treatments are no more appliable. Many studies have proved the active role of long non-coding RNAs (lncRNAs) in cancers' biology; here, the functional role of lncRNA NCK1-AS1 in HCC was identified. Gene expression in tumor tissues of HCC was evaluated by examining online databases and 88 collected HCC samples from our hospital. The interactions of miR-22-3p with NCK1-AS1 and tyrosyl-tRNA synthetase (YARS) were tested by conducting bioinformatics analysis, luciferase report, and RNA pulldown experiments. CCK-8, colony formation, flow cytometry, wound healing, transwell experiments were used to dissect the role of the NCK1-AS1/miR-22-3p/YARS axis in HCC. NCK1-AS1 was overexpressed in HCC cells and tissues. Functional assays depicted that depletion of NCK1-AS1 hampered malignant character of HCC cells. NCK1-AS1 controlled the availability of miR-22-3p, resulting in YARS upregulation. YARS was found to have a clinical value for HCC diagnosis. Moreover, rescue experiments revealed that miR-22-3p inhibition or YARS overexpression partially blocked the function of NCK1-AS1 deficiency in HCC cells. As for the downstream signaling pathway, we discovered that NCK1-AS1 activated PI3K/AKT signaling by the miR-22-3p/YARS axis. The present study verified that NCK1-AS1 could promote HCC progression via the miR-22-3p/YARS axis to activate PI3K/AKT signaling.

Molecular Docking Studies, Chemical Composition, Antioxidant, Cytotoxicity, Antibacterial and Antifungal Activities of Globularia alypum Extract

Background: Globularia alypum L. is a Mediterranean plant of the Globulariaceae family which has been used in folk medicine to cure several diseases. Different studies have been done in vitro and in vivo using diverse G. alypum extracts to understand this traditional use. Methods: In this study, Tunisian G. alypum leaf methanol extract (GAME) was chemically identified using LC-ESI-MS, then examined in vitro for its antioxidant, antibacterial, and antifungal activities. Besides, a molecular docking study was also conducted. Results: Nineteen phenolic compounds were detected, with trans-cinnamic acid (45.14%) and luteolin 7-O-glucoside (19.82%) being the dominant ones. The GAME demonstrated important antioxidant activities, especially against 2, 2-diphenyl-l-1-picrylhydrazil (DPPH) radical (IC50 = 16.1±1.1 μg.ml-1) and exhibited an anti-proliferative effect against Vero cells with (IC50 2091 ± 177 μg.ml-1). Data also reveals that the GAME inhibited the growth of oral bacteria, in particular Streptococcus oralis (MICs value ranged from 2560 to 20480 μg.ml-1). In addition, GAME has a significant antifungal action, especially against Candida albicans ATCC 90028 (MIC=2560 μg.ml-1). Docking study identified one of the major molecules (luteolin 7-O-glucoside) present in the GAME extract, displaying a good interaction with tyrosinase (docking score -9.6 kcal.mol-1) and other antibacterial (tyrosyl-tRNA synthetase, gyrase, deformylase) and antifungal (n-myristoyltransferase, chitinase) target proteins. Conclusion: This study illustrates that GAME has potent sources of antioxidants and antimicrobials useful in combating oral bacteria. Hence GAME can be of reasonable use in food technology, processing, as well as the medical field.

Synthesis and structure–activity relationship studies of mono- and bis-thiourea derivatives featuring halogenated azo dyes with antimicrobial properties

A series of mono-substituted thiourea (2a–i) and bis-substituted thiourea-azo (3a–f) derived from paracetamol derivatives were prepared from isothiocyanates with one and two equivalents of halogenated azo dyes, respectively. The incorporation of azo-derived paracetamol via thiourea moiety in 2a–i depicted excellent microbial activity on Staphylococcus aureus, and 2-(((3-((2-bromophenyl)diazenyl)-4-hydroxyphenyl)carbamothioyl)carbamoyl)phenyl acetate, 2a showed equivalent inhibition properties to the standard ampicillin (10 mm). The structure–activity relationship studies of 2a were supported via molecular docking interaction against S. aureus tyrosyl-tRNA synthetase protein which displayed a free energy interaction of −7.7 kcal/mol compared to aspirin (−6.4 kcal/mol).

A viral RNA uses conformational rearrangements of a dynamic tRNA-like structure to hijack cellular machinery

RNA viruses use multifunctional, structural elements in their untranslated regions (UTRs) to hijack cellular machinery, but detailed mechanisms are often difficult to understand due to the dynamic nature of RNA structure. We used cryo-EM to visualize a mysterious 55kDa tRNA-like structure (TLS) at the 3 end of the brome mosaic virus (BMV) genome that is recognized and aminoacylated by cellular tyrosyl-tRNA synthetase (TyrRS) and that plays critical functions in the viral life cycle. Although the BMV TLS has served as a model system of RNA structure-function relationships for decades, the structural nature of its tRNA-mimicry had remained elusive.

Synthesis, Characterization of Novel Fused Thiabenzene-1-Methyl-1-Oxide Analogs: DFT Computational, in Silico Molecular Docking Studies and Its Antibacterial Activity

The novel and fused thiabenzene-1-methyl-1-oxide analogs (5a-e) were synthesized by the reaction of Dimethylsulfoxoniummethylide (DIMSOY) and 4-alkoxy-2H-chromene-carboxaldehydes with cyclic Michel acceptors and having a leaving group at β-position. The compounds thus prepared were characterized by mass analysis, 1H and 13C NMR techniques and screened for their antibacterial activity by in vitro and in silico studies. The antibacterial activity of all the purified analogs (5a-e) were determined for Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion technique. Molecular docking studies for these compounds (5a-e) were performed against Tyrosyl-tRNA synthetase inhibitor using AutoDock. Also discussed the Density functional theory (DFT) computational studies for these compounds (5a-e). Infectious diseases caused by MDRB (multidrug resistant bacteria) are an important problem to be addressed globally. They have been among the leading causes of death, disability, growing challenges to health security and human progress, especially in developing countries for centuries. The inspiring preliminary results of these fused thisbenzene-1-methyl-1-oxide analogs (5a-e), upon further modification will produce a best drug candidate having anti-MRSA activity.

Solvent-Free Synthesis, In Vitro and In Silico Studies of Novel Potential 1,3,4-Thiadiazole-Based Molecules against Microbial Pathogens.

A new series of 1,3,4-thiadiazoles was synthesized by the reaction of methyl 2-(4-hydroxy-3-methoxybenzylidene) hydrazine-1-carbodithioate (2) with selected derivatives of hydrazonoyl halide by grinding method at room temperature. The chemical structures of the newly synthesized derivatives were resolved from correct spectral and microanalytical data. Moreover, all synthesized compounds were screened for their antimicrobial activities using Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus subtilis, Staphylococcus aureus, and Candida albicans. However, compounds 3 and 5 showed significant antimicrobial activity against all tested microorganisms. The other prepared compounds exhibited either only antimicrobial activity against Gram-positive bacteria like compounds 4 and 6, or only antifungal activity like compound 7. A molecular docking study of the compounds was performed against two important microbial enzymes: tyrosyl-tRNA synthetase (TyrRS) and N-myristoyl transferase (Nmt). The tested compounds showed variety in binding poses and interactions. However, compound 3 showed the best interactions in terms of number of hydrogen bonds, and the lowest affinity binding energy (−8.4 and −9.1 kcal/mol, respectively). From the in vitro and in silico studies, compound 3 is a good candidate for the next steps of the drug development process as an antimicrobial drug.

Anti-bacterial efficacy of bio-fabricated silver nanoparticles of aerial part of Moringa oleifera lam: Rapid green synthesis, In-Vitro and In-Silico screening

Silver nanoparticles have been extensively used in pharmaceutical sciences as therapeutic agents, including in drug delivery but their toxicity towards healthy tissue is not explored from trials. Being herb, Moringa oleifera lam (MO) is cheap, eco-friendly, non-toxic as compared to synthetic nanoparticles. The antibacterial efficacy of silver nanoparticles of aerial parts of M. oleifera lam and bactericidal mechanisms have not been explored so far. In this study, rapid biosynthesis of M. oleifera lam embedded silver nanoparticles (MONPs) was acquired using M. oleifera lam aerial parts, and characterized by using UV, Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), and X-ray diffractometer (XRD) studies. Well diffusion method was used to assess the zone of inhibition and minimum inhibitory concentration of the MONPs on Escherichia Coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi . The probable mechanism of action was accessed through molecular docking and simulation. The size of AgNPs was spherical with less than 50 nm size and showed a crystalline peak at XRD. MONPs at 250 μg/ml inhibit the growth of all the gram-positive and gram-negative bacteria with significant antibacterial affinity. The minimum inhibitory concentration of MONPs was lower in gram-positive bacteria than gram-negative bacteria. The molecular docking studies of phytoconstituents present in MO showed luteolin (−9.2 kcal/mol) and rutin (−10.1 kcal/mol) have higher binding energy with DNA gyrase & tyrosyl tRNA synthetase inhibitors respectively. Molecular simulations study confirmed that both ligands have a good stability profile by forming different bonds with active site amino acids. Bio-fabricated silver nanoparticles exhibited noteworthy gainful impacts against selected bacterial strains and could serve as a lead for future antibacterial drug discovery. • Cost effective rapid green synthesis of Moringa olifera (MO) embedded silver nanoparticles. • Antibacterial screening of MO embedded silver nanoparticles showed significant activity against bacteria. • Screening of polyphenolic compounds from MO and docking against bacterial DNA gyrase and tyrosyl tRNA synthetase. • Simulation was performed using rutin and luteolin, these ligands showed highest binding energy with 1kzn and 1jij. • Pharmacokinetics and ADME studies of selected polyphenolic compounds from MO.

Spectral, Anti-Inflammatory, Anti-Pyretic, Leishmanicidal, and Molecular Docking Studies, Against Selected Protein Targets, of a New Bisbenzylisoquinoline Alkaloid.

A new bisbenzylisoquinoline named as chondrofolinol (1) and four reported compounds (2–5) were isolated and characterized from the roots of Berberis glaucocarpa Stapf. Anti-inflammatory, anti-pyretic, and leishmanicidal studies were performed against carrageenan-induced paw edema, yeast-induced pyrexia, and the promastigotes of Leishmania tropica, respectively. The new compound significantly reduced the paw volume in carrageenan-induced paw edema and rectal temperature in yeast-induced pyrexia at 10 and 20 mg/ kg of body weight. Chondrofolinol caused almost 100% inhibition of the promastigotes of Leishmania tropica. All the compounds displayed minimal cytotoxicity against THP-1 monocytic cells. In order to ascertain the potential macromolecular targets of chondrofolinol responsible for the observed anti-inflammatory and anti-leishmanial activities, a molecular docking study was carried out on relevant protein targets of inflammation and Leishmania. Protein targets of human endoplasmic reticulum aminopeptidase 2 (ERAP2) and human matrix metalloproteinase-1 (MMP-1) for inflammation and protein targets of N-myristoyltransferase (NMT), tyrosyl-tRNA synthetase (TyrRS), and uridine diphosphate-glucose pyrophosphorylase (UGPase) for Leishmania major were selected after thorough literature search about protein targets responsible for inflammation and Leishmania major. Chondrofolinol showed excellent docking to ERAP2 and to MMP-1. The Leishmania major protein targets with the most favorable docking scores to chondrofolinol were NMT, TyrRS, and UGPase. The study indicated that bisbenzylisoquinoline and isoquinoline alkaloids possess anti-pyretic, anti-inflammatory, and anti-leishmanial properties with minimal cytotoxicity and therefore, need to be further explored for their therapeutic potential.

Precision mouse models of Yars/dominant intermediate Charcot-Marie-Tooth disease type C and Sptlc1/hereditary sensory and autonomic neuropathy type 1.

Animal models of neurodegenerative diseases such as inherited peripheral neuropathies sometimes accurately recreate the pathophysiology of the human disease, and sometimes accurately recreate the genetic perturbations found in patients. Ideally, models achieve both, but this is not always possible; nonetheless, such models are informative. Here we describe two animal models of inherited peripheral neuropathy: mice with a mutation in tyrosyl tRNA-synthetase, YarsE196K , modeling dominant intermediate Charcot-Marie-Tooth disease type C (diCMTC), and mice with a mutation in serine palmitoyltransferase long chain 1, Sptlc1C133W , modeling hereditary sensory and autonomic neuropathy type 1 (HSAN1). YarsE196K mice develop disease-relevant phenotypes including reduced motor performance and reduced nerve conduction velocities by 4months of age. Peripheral motor axons are reduced in size, but there is no reduction in axon number and plasma neurofilament light chain levels are not increased. Unlike the dominant human mutations, the YarsE196K mice only show these phenotypes as homozygotes, or as compound heterozygotes with a null allele, and no phenotype is observed in E196K or null heterozygotes. The Sptlc1C133W mice carry a knockin allele and show the anticipated increase in 1-deoxysphingolipids in circulation and in a variety of tissues. They also have mild behavioral defects consistent with HSAN1, but do not show neurophysiological defects or axon loss in peripheral nerves or in the epidermis of the hind paw or tail. Thus, despite the biochemical phenotype, the Sptlc1C133W mice do not show a strong neuropathy phenotype. Surprisingly, these mice were lethal as homozygotes, but the heterozygous genotype studied corresponds to the dominant genetics seen in humans. Thus, YarsE196K homozygous mice have a relevant phenotype, but imprecisely reproduce the human genetics, whereas the Sptlc1C133W mice precisely reproduce the human genetics, but do not recreate the disease phenotype. Despite these shortcomings, both models are informative and will be useful for future research.

Toward a treatment of antibacterial and antifungal infections: Design, synthesis and in vitro activity of novel arylhydrazothiazolylsulfonamides analogues and their insight of DFT, docking and molecular dynamic simulations

• New arylhydrazothiazolylsulfonamide derivatives 3(a-e) have been synthesized, designed and spectral characterized. • 3d displayed more potent antimicrobial activity than the antibiotics • DFT, molecular docking and dynamics simulation were performed to explore detailed binding process • Computational approach supported the in vitro results To find out effective new antibacterial agents, a series of novel aryl-hydrazothiazolyl-sulfonamide derivatives 3a-e were synthesized and well characterized by analytical and spectroscopic techniques. All the compounds were evaluated for their antibacterial and antifungal potential and the results showed excellent antimicrobial activity of 3d against all strains, especially B. cereus (MIC = 5.54 μM vs. 17.6 μM), P. aeruginosa (MIC = 7.3 μM vs.12.8 μM), E. coli (MIC = 6.4 μM vs. 21.3 μM) and C . albicans (MIC = 6.8 μM vs. 26.4 μM) in comparison to the commercial antibiotics, tetracycline and amphotericin B, respectively. The others analogues dispalyed potent to moderate antimicrobial activity. Structure-activity relationships (SARs) provides specially that incorporation of pyrimidin-2-ylsulfamoyl group at the para -phenyl position ( 3d ) is good contributor for improving the antimicrobial activity. Further, Density Functional Theory (DFT) on 3d revealed its high stability and strongest electron-donating capability. Molecular docking and dynamic simulation studies on 3d inside the active site of S. aureus tyrosyl-tRNA synthetase (PDB ID: 1JIJ), E. coli dihydropteroate synthetase (PDB ID: 1AJ0), C. albicans dihydropteroate synthetase (PDB ID: 4HOE), and C. albicans N-myristoyl transferase (PDB ID: 1IYL) showed good binding profiles with the targets proteins, in particular with 1IYL and 4HOE receptors via the involvement of hydrogen bonding and that the formed complexes were thermodynamically highly stable. Taken together, our results make 3d as a promising lead for further drugs development.

Studying Protein Tyrosine O-Sulfation in Mammalian Cells with Genetically Encoded Sulfotyrosine.

Protein tyrosine O-sulfation (PTS) is a post-translational modification (PTM) that occurs exclusively on secreted and membrane-bound proteins. It participates in a wide range of important biological processes and is involved in the development of many diseases. The biomedical importance of PTS can only be fully unveiled when the right chemical/biological tools are available. This article outlines the steps for using an engineered Escherichia coli tyrosyl-tRNA synthetase to genetically encode sulfotyrosine (sTyr) for biological investigations of PTS in mammalian cells. Two basic protocols are described to demonstrate this methodology, including the site-specific incorporation of sTyr into eGFP protein in HEK293T cells and the functional study of an sTyr-containing CXCR4 protein using a calcium mobilization assay. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Site-specific incorporation of sTyr into eGFP Basic Protocol 2: Functional study of site-specifically sulfated CXCR4.

Progress in Incorporation of Non-canonical Amino Acid Based on Archaeal Tyrosyl-tRNA Synthetase

Progress in Incorporation of Non-canonical Amino Acid Based on Archaeal Tyrosyl-tRNA Synthetase

Interplay of substrate polymorphism and conformational plasticity of Plasmodium tyrosyl-tRNA synthetase

Aminoacyl-tRNA synthetases are an indispensable component of ribosomal protein translational machinery and Plasmodium Tyrosyl-tRNA synthetase (PfTyrRS) is a validated drug target. This manuscript illustrates the dynamic conformational landscape of PfTyrRS in the context of substrate binding. Molecular dynamics simulations of PfTyrRS in the presence and absence of ligand show conformational heterogeneity for both the protein and the bound ligand. Diverse conformations for the evolutionarily conserved ATP binding motif (KMSKS) have been observed in both apo- and holo PfTyrRS. Further, the presented attributes of the tyrosyl-adenylate conformational sub-states in situ along with their implications on the strength of intermolecular interactions would be a pertinent benchmark for molecular design studies. In addition, an analysis of the ligand hydration pattern foregrounds the structurally conserved water-mediated inter-molecular interactions. The quantitative assessment of the conformational landscape, based on the fluctuations of the distance between the ligand binding pockets, of apo-PfTyrRS and holo-PfTyrRS highlights the nature of diversity in conformational sampling for the two cases. Evidently, the holo-PfTyrRS adopts a rather compact conformation compared to the apo-PfTyrRS. An intriguing asymmetry in the dynamics of the two monomers is contextualized with the functional asymmetry of the symmetrically dimeric PfTyrRS. Importantly, the network of non-bonded contacts in the apo- and holo- simulated ensembles has been analyzed. The graph-theoretic analysis-based novel insights concerning the nature of information flow as a function of ligation state would prove valuable in understanding PfTyrRS functions. The results presented here contend that understanding allostery in PfTyrRS is essential to astutely design structure-based inhibitors.

The recurrent missense mutation p.(Arg367Trp) in YARS1 causes a distinct neurodevelopmental phenotype

Pathogenic variants in aminoacyl-tRNA synthetases (ARS1) cause a diverse spectrum of autosomal recessive disorders. Tyrosyl tRNA synthetase (TyrRS) is encoded by YARS1 (cytosolic, OMIM*603,623) and is responsible of coupling tyrosine to its specific tRNA. Next to the enzymatic domain, TyrRS has two additional functional domains (N-Terminal TyrRSMini and C-terminal EMAP-II-like domain) which confer cytokine-like functions. Mutations in YARS1 have been associated with autosomal-dominant Charcot-Marie-Tooth (CMT) neuropathy type C and a heterogenous group of autosomal recessive, multisystem diseases. We identified 12 individuals from 6 families with the recurrent homozygous missense variant c.1099C > T;p.(Arg367Trp) (NM_003680.3) in YARS1. This variant causes a multisystem disorder with developmental delay, microcephaly, failure to thrive, short stature, muscular hypotonia, ataxia, brain anomalies, microcytic anemia, hepatomegaly, and hypothyroidism. In silico analyses show that the p.(Arg367Trp) does not affect the catalytic domain responsible of enzymatic coupling, but destabilizes the cytokine-like C-terminal domain. The phenotype associated with p.(Arg367Trp) is distinct from the other biallelic pathogenic variants that reside in different functional domains of TyrRS which all show some common, but also divergent clinical signs [(e.g., p.(Phe269Ser)—retinal anomalies, p.(Pro213Leu)/p.(Gly525Arg)—mild ID, p.(Pro167Thr)—high fatality)]. The diverse clinical spectrum of ARS1-associated disorders is related to mutations affecting the various non-canonical domains of ARS1, and impaired protein translation is likely not the exclusive disease-causing mechanism of YARS1- and ARS1-associated neurodevelopmental disorders.Key messagesThe missense variant p.(Arg367Trp) in YARS1 causes a distinct multisystem disorder.p.(Arg367Trp) affects a non-canonical domain with cytokine-like functions.Phenotypic heterogeneity associates with the different affected YARS1 domains.Impaired protein translation is likely not the exclusive mechanism of ARS1-associated disorders.

3-(5-(1H-imidazol-1-yl) pent-1-en-1-yl)-9-ethyl-9H-carbazole: synthesis, characterization (IR, NMR), DFT, antimicrobial-antioxidant activities and docking study

3-(5-(1H-imidazol-1-yl) pent-1-en-1-yl)-9-ethyl-9H-carbazole called as compound 1 was synthesized and characterized by proton and carbon-13 nuclear magnetic resonance (1H- and 13C- NMR) and Fourier transform infrared (FTIR) spectroscopic methods. Density Functional Theory/Becke, 3-parameter (DFT/B3LYP), for compound 1 were performed with 6-311++G(d,p) method. Optimized geometry, frontier molecular orbitals (HOMO; highest occupied molecular orbital; LUMO: lowest unoccupied molecular orbital), IR and NMR parameters of compound 1 were obtained. The evaluations reveal that the calculation results support the experimental results. In addition, the antimicrobial (a microwell dilution method) and antioxidant activities (2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) ferric ion reducing antioxidant power (FRAP) of compound 1 were evaluated. According to the results obtained, it showed higher antimicrobial activity (Minimal inhibition concentration (MIC): 78.12 µg/mL) against B. subtilis subsp. Spizizenii. Morever, molecular docking studies were carried out to investigate the interactions of an antimicrobial agent on some important enzymes played important roles in nucleic acid (Deoxyribo nucleic acid (DNA) synthesis, cell wall synthesis, protein synthesis, and metabolism etc. The compound 1 was strongly bound to tyrosyl-tRNA synthetase enzyme (binding energy: −11.18 and Ki: 6.37 nM) and Beta-Ketoacyl-Acp Synthase III enzyme (binding energy: −10.29 and Ki: 28.47 nM). Communicated by Ramaswamy H. Sarma

Role of two aminoacyl-tRNA synthetase associated proteins (Endothelial Monocyte Activating Polypeptides 1 and 2) of Leishmania donovani in chemotaxis of human monocytes.

Visceral leishmaniasis is caused by the protozoan parasite Leishmania donovani. It is a fatal form of leishmaniasis prevalent in Indian subcontinent. Since there are no human licensed vaccines available for leishmaniasis, chemotherapeutic drugs remain the only means for combating parasitic infections. We have earlier identified a total of 26 amino-acyl tRNA synthetases (aaRS) along with five stand-alone editing domains and two aaRS-associated proteins in Leishmania donovani. In addition to their canonical role of tRNA aminoacylation, aaRS have been involved in novel functions by acquiring novel domains during evolution. The aaRS-associated proteins have been reported to be analogous to a human cytokine, EMAP II, as they possess a modified version of the heptapeptide motif responsible for the cytokine activity. In this manuscript, we report the characterization of two L. donovani aminoacyl-tRNA synthetase associated proteins which showed a human chemokine like activity. Both the proteins, L. donovani EMAP-1 and EMAP-2, possess a modified form of the heptapeptide motif, which is responsible for cytokine activity in human EMAP-2. LdEMAP-1 and LdEMAP-2 were cloned, expressed, and purified. Both LdEMAP-1 and LdEMAP-2 proteins in the promastigote stage were found to be localized in cytoplasm as confirmed by immunofluorescence. In case of L. donovani infected human THP-1 derived macrophages, secretion of LdEMAP-1 and LdEMAP-2 proteins in the cytosol of the macrophages was observed. The role of LdEMAP-1 and LdEMAP-2 in the aminoacylation of rLdTyrRS was also tested and LdEMAP-2 but not LdEMAP-1 increased the rate of aminoacylation of tyrosyl tRNA synthetase (rLdTyrRS). L. donovani EMAP-1 and EMAP-2 proteins managed to exhibit the capability of attracting human origin cells as determined by chemotaxis assay, and also were able to induce the secretion of cytokines from macrophages like their human counterpart (EMAP II). Our working hypothesis is that both of these proteins might be involved in helping the parasite to establish the infection within the host.

Antileishmanial Potential of Berberine Alkaloids From Berberis glaucocarpa Roots: Molecular Docking Suggests Relevant Leishmania Protein Targets

Leishmaniases are a spectrum of poverty-linked neglected parasitic diseases that are endemic in 88 countries around the globe and affect millions of people every year. Currently available chemotherapeutic options are inadequate due to side effects, high cost, prolonged treatment, and parasite resistance. Thus, there is an existing need to develop new potent and safer leishmanicidal drugs. Considering the folkloric antiulcer and leishmanicidal use of the genus Berberis and its alkaloids, 5 reported alkaloids, namely berberine (1), palmatine (2), columbamine (3), 8-trichloromethyldihydroberberine (4), and jatrorrhizine (5), were isolated from the roots of Berberis glaucocarpa using classical (column and preparative chromatography) and modern isolation techniques (Sephadex LH-20). Their structures were elucidated and established from 1D and 2D spectroscopic data. The isolated alkaloids displayed excellent antileishmanial potential with IC50 values ranging from 1.50 to 2.56 µM: 1 (1.50 ± 0.53 µM), 2 (2.31 ± 0.37 µM), 3 (2.56 ± 0.48 µM), 4 (1.40 ± 0.90 µM), 5 (2.44 ± 1.34 µM). While the IC50 value for the standard drug (Amphotericin-B) was found to be 1.08 ± 0.95 µM. All of the isolated alkaloids displayed excellent antileishmanial potential as well as minimal cytotoxicity against THP-1 monocytic cells. Molecular docking analysis has revealed Leishmania N-myristoyl transferase, methionyl-tRNA synthetase, pteridine reductase 1, oligopeptidase B, tyrosyl-tRNA synthetase, and/or glycerol-3-phosphate dehydrogenase to be potential protein targets for the alkaloids.

Crystal structure of Nanoarchaeum equitans tyrosyl-tRNA synthetase and its aminoacylation activity toward tRNATyr with an extra guanosine residue at the 5ʹ-terminus

tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5ʹ-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5ʹ-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 Å resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5ʹ-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.

Two Novel Variants in YARS2 Gene Are Responsible for an Extended MLASA Phenotype with Pancreatic Insufficiency

Pathogenic variants in the mitochondrial tyrosyl-tRNA synthetase gene (YARS2) were associated with myopathy, lactic acidosis, and sideroblastic anemia (MLASA). However, patients can present mitochondrial myopathy, with exercise intolerance and muscle weakness, leading from mild to lethal phenotypes. Genes implicated in mtDNA replication were studied by Next Generation Sequencing (NGS) and whole exome sequence with the TruSeq Rapid Exome kit (Illumina, San Diego, CA, USA). Mitochondrial protein translation was studied following the Sasarman and Shoubridge protocol and oxygen consumption rates with Agilent Seahorse XF24 Analyzer Mitostress Test, (Agilent, Santa Clara, CA, USA). We report two siblings with two novel compound heterozygous pathogenic variants in YARS2 gene: a single nucleotide deletion in exon 1, c.314delG (p.(Gly105Alafs*4)), which creates a premature stop codon in the amino acid 109, and a single nucleotide change in exon 5 c.1391T>C (p.(Ile464Thr)), that cause a missense variant in amino acid 464. We demonstrate the pathogenicity of these new variants associated with reduced YARS2 mRNA transcript, reduced mitochondrial protein translation and dysfunctional organelle function. These pathogenic variants are responsible for late onset MLASA, herein accompanied by pancreatic insufficiency, observed in both brothers, clinically considered as Pearson’s syndrome. Molecular study of YARS2 gene should be considered in patients presenting Pearson’s syndrome characteristics and MLASA related phenotypes.