scholarly journals Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Sanya Chadha ◽  
N. Arjunreddy Mallampudi ◽  
Debendra K. Mohapatra ◽  
Rentala Madhubala
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Essential Gene ◽  
Protozoan Parasite ◽  
Protein Translation ◽  
Trna Synthetase ◽  
Parasite Growth ◽  
Coding Sequences ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target.

scholarly journals Aminoacyl tRNA Synthetases as Malarial Drug Targets: A Comparative Bioinformatics Study

2018 ◽  
Author(s):  
Dorothy Wavinya Nyamai ◽  
Özlem Tastan Bishop
Keyword(s):  
Amino Acid ◽  
Drug Targets ◽  
Motif Discovery ◽  
Protein Translation ◽  
Trna Synthetase ◽  
Potential Drug ◽  
Multiple Sequence ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Potential Drug Targets

AbstractTreatment of parasitic diseases has been challenging due to the development of drug resistance by parasites, and thus there is need to identify new class of drugs and drug targets. Protein translation is important for survival of plasmodium and the pathway is present in all the life cycle stages of the plasmodium parasite. Aminoacyl tRNA synthetases are primary enzymes in protein translation as they catalyse the first reaction where an amino acid is added to the cognate tRNA. Currently, there is limited research on comparative studies of aminoacyl tRNA synthetases as potential drug targets. The aim of this study is to understand differences between plasmodium and human aminoacyl tRNA synthetases through bioinformatics analysis. Plasmodium falciparum, P. fragile, P. vivax, P. ovale, P. knowlesi, P. bergei, P. malariae and human aminoacyl tRNA synthetase sequences were retrieved from UniProt database and grouped into 20 families based on amino acid specificity. Despite functional and structural conservation, multiple sequence analysis, motif discovery, pairwise sequence identity calculations and molecular phylogenetic analysis showed striking differences between parasite and human proteins. Prediction of alternate binding sites revealed potential druggable sites in PfArgRS, PfMetRS and PfProRS at regions that were weakly conserved when compared to the human homologues. These differences provide a basis for further exploration of plasmodium aminoacyl tRNA synthetases as potential drug targets.

scholarly journals Expression of genes for selected plant aminoacyl-tRNA synthetases in the abiotic stress

2020 ◽  
Vol 80 (1) ◽  
Author(s):  
Jurica Baranašić ◽  
Anita Mihalak ◽  
Ita Gruić-Sovulj ◽  
Nataša Bauer ◽  
Jasmina Rokov-Plavec
Keyword(s):  
Gene Expression ◽  
Stress Responses ◽  
Environmental Changes ◽  
Cadmium Stress ◽  
Protein Translation ◽  
Trna Synthetase ◽  
Stress Conditions ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Global Translation

Plants, as sessile organisms, have evolved intricate mechanisms to adapt to various environmental changes and challenges. Considering that various types of stress trigger significant decrease in global translation rates we examined stress-related expression of aminoacyl-tRNA synthetases (aaRSs), enzymes that participate in the first step of protein translation. We have analyzed promoters of genes encoding cytosolic seryl-tRNA synthetase (SerRS), cytosolic aspartyl-tRNA synthetase (AspRS) and cytosolic cysteinyl-tRNA synthetase (CysRS) in Arabidopsis thaliana L., and examined SerRS, AspRS and CysRS gene expression in the seedlings exposed to different abiotic stressors. Although global translation levels are repressed by stress, our results show that plant aaRSs expression is not decreased by osmotic, salt and heavy metal/cadmium stress. Moreover, during exposure to stress conditions we detected increased AspRS and CysRS transcript levels. SerRS gene expression did not change, however participation of SerRS in stress response could be regulated at the protein level. Expression of the examined aaRS genes in stress correlated well with the length of their predicted promoters and a number of available binding sites for the stress related transcription factors. It thus appears that during the stress it is important to keep steady state levels of aaRSs for translation of specific stress related mRNAs and furthermore to rapidly continue with translation when stress conditions cease. Importantly, increased levels of plant aaRSs during stress may serve as a pool of aaRS proteins that can participate directly in stress responses through their noncanonical activities.

scholarly journals Plant-Specific Domains and Fragmented Sequences Imply Non-Canonical Functions in Plant Aminoacyl-tRNA Synthetases

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1056
Author(s):  
Yusuke Saga ◽  
Moeka Kawashima ◽  
Shiho Sakai ◽  
Kaori Yamazaki ◽  
Misato Kaneko ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Protein Translation ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Genome Wide ◽  
A Genome ◽  
Specific Manner ◽  
Number Of Genes ◽  
Genome Wide Search

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein translation. In addition, numerous aaRSs (mostly in vertebrates) have also been discovered to possess a range of non-canonical functions. Very few studies have been conducted to elucidate or characterize non-canonical functions of plant aaRSs. A genome-wide search for aaRS genes in Arabidopsis thaliana revealed a total of 59 aaRS genes. Among them, asparaginyl-tRNA synthetase (AsnRS) was found to possess a WHEP domain inserted into the catalytic domain in a plant-specific manner. This insertion was observed only in the cytosolic isoform. In addition, a long stretch of sequence that exhibited weak homology with histidine ammonia lyase (HAL) was found at the N-terminus of histidyl-tRNA synthetase (HisRS). This HAL-like domain has only been seen in plant HisRS, and only in cytosolic isoforms. Additionally, a number of genes lacking minor or major portions of the full-length aaRS sequence were found. These genes encode 14 aaRS fragments that lack key active site sequences and are likely catalytically null. These identified genes that encode plant-specific additional domains or aaRS fragment sequences are candidates for aaRSs possessing non-canonical functions.

scholarly journals Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bingyi Chen ◽  
Siting Luo ◽  
Songxuan Zhang ◽  
Yingchen Ju ◽  
Qiong Gu ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Domain ◽  
Trna Synthetase ◽  
Rational Drug Design ◽  
Class I ◽  
Binding Assays ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Rational Drug ◽  
Trna Binding

AbstractThe polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with l-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

2012 ◽  
Vol 443 (2) ◽  
pp. 477-484 ◽  
Author(s):  
Min Tan ◽  
Wei Yan ◽  
Ru-Juan Liu ◽  
Meng Wang ◽  
Xin Chen ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Trna Synthetase ◽  
Modular Construction ◽  
Aquifex Aeolicus ◽  
Trna Synthetases ◽  
Naturally Occurring ◽  
Aminoacyl Trna Synthetases ◽  
Editing Domain ◽  
Editing Activity ◽  
Shed Light

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.

scholarly journals Measuring the tolerance of the genetic code to altered codon size

2021 ◽  
Author(s):  
E. DeBenedictis ◽  
D. Söll ◽  
K. Esvelt
Keyword(s):  
Genetic Code ◽  
Protein Translation ◽  
Single Amino Acid ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases ◽  
Triplet Codon ◽  
Orthogonal Set ◽  
Expanded Genetic Code ◽  
Primordial Earth

SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

scholarly journals Influence of Antisynthetase Antibodies Specificities on Antisynthetase Syndrome Clinical Spectrum Time Course

2019 ◽  
Vol 8 (11) ◽  
pp. 2013 ◽  
Author(s):  
Cavagna ◽  
Trallero-Araguás ◽  
Meloni ◽  
Cavazzana ◽  
Rojas-Serrano ◽  
...  
Keyword(s):  
Time Course ◽  
Clinical Presentation ◽  
Reference Group ◽  
Trna Synthetase ◽  
Antisynthetase Syndrome ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Antisynthetase Antibodies ◽  
Clinical Triad

Antisynthetase syndrome (ASSD) is a rare clinical condition that is characterized by the occurrence of a classic clinical triad, encompassing myositis, arthritis, and interstitial lung disease (ILD), along with specific autoantibodies that are addressed to different aminoacyl tRNA synthetases (ARS). Until now, it has been unknown whether the presence of a different ARS might affect the clinical presentation, evolution, and outcome of ASSD. In this study, we retrospectively recorded the time of onset, characteristics, clustering of triad findings, and survival of 828 ASSD patients (593 anti-Jo1, 95 anti-PL7, 84 anti-PL12, 38 anti-EJ, and 18 anti-OJ), referring to AENEAS (American and European NEtwork of Antisynthetase Syndrome) collaborative group’s cohort. Comparisons were performed first between all ARS cases and then, in the case of significance, while using anti-Jo1 positive patients as the reference group. The characteristics of triad findings were similar and the onset mainly began with a single triad finding in all groups despite some differences in overall prevalence. The “ex-novo” occurrence of triad findings was only reduced in the anti-PL12-positive cohort, however, it occurred in a clinically relevant percentage of patients (30%). Moreover, survival was not influenced by the underlying anti-aminoacyl tRNA synthetase antibodies’ positivity, which confirmed that antisynthetase syndrome is a heterogeneous condition and that antibody specificity only partially influences the clinical presentation and evolution of this condition.

Aminoacyl-tRNA synthetases of Halobacterium cutirubrum: preliminary fractionation studies

1970 ◽  
Vol 48 (8) ◽  
pp. 944-946 ◽  
Author(s):  
E. Griffiths
Keyword(s):  
Ionic Strength ◽  
Gel Filtration ◽  
Halophilic Bacterium ◽  
Trna Synthetase ◽  
Partial Purification ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Fractionation Procedure ◽  
The Stability ◽  
Low Ionic Strength

The stability, in solutions of low ionic strength, of aminoacyl-tRNA synthetases from the extremely halophilic bacterium Halobacterium cutirubrum was studied as a preliminary to their fractionation. The enzymes differed considerably in their sensitivity to such solutions. Conditions were found where reactivation from the salt-free and inactive state could be achieved. Removal of both K+ and Mg2+ together generally resulted in better stability than the removal of K+ alone. A low temperature (4°) was also important for stability in buffers of low ionic strength. In some cases the L-amino acid substrates afforded protection against inactivation in the salt-free state. Gel filtration in low ionic strength medium was found to work well as a fractionation procedure; a partial purification of phenylalanyl-tRNA synthetase was effected in this way. The use of other conventional protein fractionation procedures is now possible.

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