scholarly journals Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases

2020 ◽  
Vol 295 (41) ◽  
pp. 13981-13993
Author(s):  
Charlotta Preger ◽  
Edvard Wigren ◽  
Elena Ossipova ◽  
Carolyn Marks ◽  
Johan Lengqvist ◽  
...  
Keyword(s):  
Cell Types ◽  
Trna Synthetase ◽  
Future Research ◽  
Binding Assays ◽  
Single Chain ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Single Chain Fragment Variable ◽  
Endogenous Protein ◽  
Purified Antigen

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.

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 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 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.

scholarly journals Polyribosomal and particulate distribution of lysyl- and phenylalanyl-transfer ribonucleic acid synthetases

1974 ◽  
Vol 143 (1) ◽  
pp. 191-195 ◽  
Author(s):  
Gale Moline ◽  
Arnold Hampel ◽  
M. Duane Enger
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Trna Synthetase ◽  
Differential Sensitivity ◽  
Synthetase Activity ◽  
Ribosomal Subunits ◽  
Ovary Cells ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Particulate Distribution

1. Only two aminoacyl-tRNA synthetases from Chinese hamster ovary cells are found associated with ribosomes and polyribosomes. 2. Phenylalanyl-tRNA synthetase activity is found with the 60S subunit, 80S monoribosome and individual polyribosomes. An additional 15S form of the enzyme is also seen. 3. Lysyl-tRNA synthetase activity is found in a form of about 20S and associated with ribosomal subunits and polyribosomes. The ribosomal subunits having lysyl-tRNA synthetase activity are about 6S larger than the bulk of the ribosomal subunits. 4. The lysyl- and phenylalanyl-tRNA synthetases found in different complexes have differential sensitivity to EDTA and centrifugation properties.

scholarly journals Role of Nuclear Pools of Aminoacyl-tRNA Synthetases in tRNA Nuclear Export

2001 ◽  
Vol 12 (5) ◽  
pp. 1381-1392 ◽  
Author(s):  
Abul K. Azad ◽  
David R. Stanford ◽  
Srimonti Sarkar ◽  
Anita K. Hopper
Keyword(s):  
Nuclear Localization ◽  
Nuclear Export ◽  
Trna Synthetase ◽  
Nuclear Localization Sequence ◽  
Sequence Alignments ◽  
Trna Aminoacylation ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Localization Sequence ◽  
Nuclear Location

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export ( Lund and Dahlberg, 1998 ; Sarkar et al., 1999 ; Grosshans et al., 2000a ) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.

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