Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target.

BACKGROUND: Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis. METHODOLOGY: Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human. CONCLUSIONS: Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 µM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 µM). Borrelidin inhibited the promastigotes (IC50: 21 µM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts.

Reinvestigation of aminoacyl-tRNA synthetase core complex by affinity purification-mass spectrometry reveals TARSL2 as a potential member of the complex.

Twenty different aminoacyl-tRNA synthetases (ARSs) link each amino acid to their cognate tRNAs. Individual ARSs are also associated with various non-canonical activities involved in neuronal diseases, cancer and autoimmune diseases. Among them, eight ARSs (D, EP, I, K, L, M, Q and RARS), together with three ARS-interacting multifunctional proteins (AIMPs), are currently known to assemble the multi-synthetase complex (MSC). However, the cellular function and global topology of MSC remain unclear. In order to understand the complex interaction within MSC, we conducted affinity purification-mass spectrometry (AP-MS) using each of AIMP1, AIMP2 and KARS as a bait protein. Mass spectrometric data were funneled into SAINT software to distinguish true interactions from background contaminants. A total of 40, 134, 101 proteins in each bait scored over 0.9 of SAINT probability in HEK 293T cells. Complex-forming ARSs, such as DARS, EPRS, IARS, Kars, LARS, MARS, QARS and RARS, were constantly found to interact with each bait. Variants such as, AIMP2-DX2 and AIMP1 isoform 2 were found with specific peptides in KARS precipitates. Relative enrichment analysis of the mass spectrometric data demonstrated that TARSL2 (threonyl-tRNA synthetase like-2) was highly enriched with the ARS-core complex. The interaction was further confirmed by coimmunoprecipitation of TARSL2 with other ARS core-complex components. We suggest TARSL2 as a new component of ARS core-complex.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix.

The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P4(1)2(1)2 or P4(3)2(1)2 space group and consist of one monomer per asymmetric unit.

Crystallization and preliminary crystallographic studies of putative threonyl-tRNA synthetases from Aeropyrum pernix and Sulfolobus tokodaii.

Threonyl-tRNA synthetase (ThrRS) plays an essential role in protein synthesis by catalyzing the aminoacylation of tRNA(Thr) and editing misacylation. ThrRS generally contains an N-terminal editing domain, a catalytic domain and an anticodon-binding domain. The sequences of the editing domain in ThrRSs from archaea differ from those in bacteria and eukaryotes. Furthermore, several creanarchaea including Aeropyrum pernix K1 and Sulfolobus tokodaii strain 7 contain two genes encoding either the catalytic or the editing domain of ThrRS. To reveal the structural basis for this evolutionary divergence, the two types of ThrRS from the crenarchaea A. pernix and S. tokodaii have been overexpressed in Eschericha coli, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected and the structure of a selenomethionine-labelled A. pernix type-1 ThrRS crystal has been solved using the MAD method.

Threonyl-tRNA synthetase.

Threonine contributes to the solubility and reactivity of proteins by its hydroxy group as well as to the formation and stability of the hydrophobic core of proteins by its methyl group. One may assume that the use of this bifunctional and simply structured amino acid was established early in evolution. Whereas the catalytic pathway of threonine activation and transfer into protein does not deviate essentially from those catalyzed by other aminoacyl-tRNA synthetases, the enzyme specific for threonine exhibits several interesting individual properties: its biosynthesis is regulated by feedback mechanisms, it can be selectively inhibited (out of twenty aminoacyl-tRNA synthetases) by the antibiotic borrelidin, and it can be a target for autoantibodies, thus being involved in the course of autoimmune diseases. The enzyme has been isolated from more than ten organisms showing a dimeric nature and molecular masses between 110 and 220 kDa. Additionally, in several of these cases, the gene of threonyl-tRNA synthetase has been localized, cloned and sequenced, exhibiting proteins of 400 to 800 amino acids chain length. More interesting facts can be expected from future research ranging from chemistry and molecular biology to medicine, e.g. by elucidation of the three dimensional structures of threonyl-tRNA synthetases and of their antigenic epitopes, possibly followed by therapeutic use of less antigenic mutant proteins.

Presence of role of the 5SrRNA-L5 protein complex (5SRNP) in the threonyl- and histidyl-tRNA synthetase complex in rat liver cytosol.

A complex containing Thr-RS and His-RS was purified about 1000 to 2000-fold from rat liver cytosol by successive column chromatographies on Sephadex G-200, Phenyl-Sepharose CL-4B, and tRNA-Sepharose. The ratio of the specific activity of Thr-RS and His-RS was relatively constant throughout the purification steps, suggesting that the two synthetases were co-purified as a complex. Chromatographic analyses of the tRNA-Sepharose fraction by Sephadex G-150 column chromatography showed the presence of a hybrid form of the Thr-RS monomer and the His-RS monomer in addition to dimer forms of both enzymes from the pattern of activity of both enzymes. The monomer form of Thr-RS showed high activity comparable to the dimer form and the monomer form of His-RS showed definite activity. An association form of Thr-RS and His-RS dimers was detected by Sephadex G-200 chromatography of rat liver cytosol. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 55SrRNA blot analysis of the tRNA-Sepharose fraction using an antibody against ribosomal protein L5, showed the presence of ribosomal protein L5 in this fraction. These findings suggest that the presence of a 5SRNA-L5 protein complex (5SRNP) in the Thr-RS and His-RS complex. 5SRNP enhanced the activity of Thr-RS in a freshly prepared tRNA-Sepharose fraction. It also enhanced the activity of the rat liver cytosol for the attachment of [3H]threonine to endogenous tRNA. This activity was inhibited by an antibody against protein L5, and the inhibition was reversed by addition of 5SRNP. These results indicate that 5SRNP plays a role as a positive effector of Thr-RS in the complex.

Threonyl-tRNA synthetase from Thermus thermophilus: purification and some structural and kinetic properties.

Threonyl-tRNA synthetase (ThrRS) has been isolated from an extreme thermophile Thermus thermophilus strain HB8. The enzyme was purified to electrophoretic homogeneity by combinations of column chromatographies on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel and HA-Ultrogel. Seventeen mg of purified enzyme were obtained from 1 kg of biomass. In parallel, purified aspartyl- and phenylalanyl-tRNA synthetases were obtained. The purified ThrRS is composed of two identical subunits with a molecular mass of about 77,000 (virtually the same as E coli ThrRS). The N-terminal sequence has been determined. The homology between the first 45 amino acid residues of ThrRS from T thermophilus and E coli is about 29%. A comparative study of tRNA(Thr) charging by ThrRS from E coli and T thermophilus reveals a similar efficiency of the reaction in both homologous systems. This efficiency remains unchanged for aminoacylation of tRNA(Thr) from T thermophilus by the heterologous ThrRS from E coli, but decreases 700 times for aminoacylation of E coli tRNA(Thr) by ThrRS from T thermophilus.

Purification of aminoacyl-tRNA synthetase kinase activities associated with threonyl- and tyrosyl-tRNA synthetases isolated from Bom:NMRI mouse liver.

Aminoacyl-tRNA synthetase kinase activities and aminoacyl-tRNA synthetase activities prepared from postribosomal supernatants of Bom:NMRI mouse liver comigrated through the following purification steps: 1 a) gel filtration on Sephadex G-200, 1 b) chromatofocusing, 2) affinity chromatography on immobilised total tRNA (mixture of tRNA's, specific for all aminoacyl-tRNA synthetases) and 3) affinity chromatography on immobilised tRNA, specific for each of threonyl- and tyrosyl-tRNA synthetases. The purification factors for threonyl- and tyrosyl-tRNA synthetases were about 17000x. The purified synthetases showed only one protein band following sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified threonyl- and tyrosyl-tRNA synthetase proteins were found also to possess threonyl- and tyrosyl-tRNA synthetase kinase activities, respectively. The purification of tRNA(Thr) and tRNA(Tyr) as well as a method for the renaturation and identification of threonyl- and tyrosyl-tRNA synthetase activities in protein bands obtained by SDS PAGE are described.

Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence.

In Bacillus subtilis, two genes, thrS and thrZ, encode distinct threonyl-tRNA synthetase enzymes. Normally, only the thrS gene is expressed. Here we show that either gene, thrS or thrZ, is sufficient for normal cell growth and sporulation. Reducing the intracellular ThrS protein concentration induces thrZ expression in a dose-compensatory manner. Starvation for threonine simultaneously induces thrZ and stimulates thrS expression. The 5'-leader sequences of thrS and thrZ contain, respectively, one and three transcription terminators preceded by a conserved sequence. We show that this sequence is essential for the regulation of thrS via a transcriptional antitermination mechanism. We propose that both genes, thrS and thrZ, are regulated by the same mechanism such that the additional regulatory domains present before thrZ account for its non-expression. In contrast to Escherichia coli, structurally similar regulatory domains, i.e. the consensus sequence preceding a terminator structure, are found in the leader regions of most aminoacyl-tRNA synthetase genes of Gram-positive bacteria. This suggests that they are regulated by a common mechanism.

Crystals of threonyl-tRNA synthetase from Thermus thermophilus. Preliminary crystallographic data.

Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant. The crystals are very stable and diffract to at least 2.4 A. The crystals belong to space group P2(1)2(1)2(1) with cell parameters a = 61.4 A, b = 156.1 A, c = 177.3 A.

An anticodon change switches the identity of E. coli tRNA(mMet) from methionine to threonine.

Recent evidence indicates that the anticodon may often play a crucial role in selection of tRNAs by aminoacyl-tRNA synthetases. In order to quantitate the contribution of the anticodon to discrimination between cognate and noncognate tRNAs by E. coli threonyl-tRNA synthetase, derivatives of the E. coli elongator methionine tRNA (tRNA(mMet)) containing wild type and threonine anticodons have been synthesized in vitro and assayed for threonine acceptor activity. Substitution of the threonine anticodon GGU for the methionine anticodon CAU increased the threonine acceptor activity of tRNA(mMet) by four orders of magnitude while reducing methionine acceptor activity by an even greater amount. These results indicate that the anticodon is the major element which determines the identity of both threonine and methionine tRNAs.

Phosphorylation of threonyl- and seryl-tRNA synthetase by cAMP-dependent protein kinase. A possible role in the regulation of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) synthesis.

Threonyl-tRNA synthetase has been shown to be phosphorylated in reticulocytes (Dang, C. V., Tan, E. M., and Traugh, J. A., (1988) FASEB J. 2, 2376-2379). Upon incubation of reticulocytes with 8-bromo-cAMP, phosphorylation of threonyl-tRNA synthetase is stimulated approximately 2-fold, an increase similar to that observed with ribosomal protein S6. To analyze the effects of phosphorylation on activity, threonyl-tRNA synthetase has been purified to apparent homogeneity from rabbit reticulocytes utilizing a four-step purification procedure with the simultaneous purification of seryl-tRNA synthetase. Both synthetases are phosphorylated in vitro by the cAMP-dependent protein kinase. Prior to phosphorylation, the two synthetases produce significant amounts of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) in the presence of the cognate amino acid and ATP, with activities comparable to that of lysyl-tRNA synthetase. Phosphorylation has no effect on aminoacylation, but an increase in Ap4A synthesis of up to 6-fold is observed with threonyl-tRNA synthetase and 2-fold with seryl-tRNA synthetase. Thus, cAMP-mediated phosphorylation of specific aminoacyl-tRNA synthetases appears to be a potential mode of regulation of Ap4A synthesis in mammals.

[Threonyl-tRNA synthase from rabbit reticulocytes: a reversible transition from the RNA-non-binding to the RNA-binding form]. / Treonil-tRNK-sintetaza retikulotsitov krolika: obratimyi perekhod iz RNK-nesviazyvaiushchei v RNK-sviazyvaiushchuiu formu.

A simple three-step procedure was used to isolate threonyl-tRNA synthetase of rabbit reticulocytes which is in a ribosome-free extract in the RNA-non-binding form. According to SDS electrophoresis, the enzyme has a molecular weight of 86 000 Da and is heterogeneous by isoelectric point; pI of the major component is near 6.2. Threonyl-tRNA synthetase is capable of interacting with a high molecular weight RNA (E. coli rRNA). Thus, in the course of purification threonyl-tRNA synthetase passes from the RNA-non-binding to the RNA-binding form. This transition was shown to be reversible.

Threonyl-tRNA synthetase from bovine liver. Purification and kinetic properties in the ATP-pyrophosphate exchange reaction.

Threonyl-tRNA synthetase (E.C. 6.1.1.3) from bovine liver has been purified to near homogeneity approximately 500 fold with a recovery of 48%. Two bands of molecular weight 90,000 and 82,000 respectively, were obtained by sodium dodecyl sulfate gel electrophoresis. The enzyme has an isoelectric point of 5.2 by polyacrylamide containing ampholine gel electrophoresis. Optimum assay conditions and apparent Km values have been determined in the ATP-PPi exchange reaction. Effects of divalent cations and diamine in substitution to Mg2+ and effects of sulfhydryl reagents on ATP-PPi exchange activity have also been observed.

Threonyl-tRNA synthetase from bovine liver: purification and kinetic properties in the ATP-pyrophosphate exchange reaction

Treonil-RNA sintetase (E.C. 6.1.1.3) foi purificada quase à homogeneidade de fígado bovino cerca de 500 vezes com um rendimento de 48%. Duas bandas de pesos moleculares 90.000 e 82.000, respectivamente, foram obtidas por eletroforese em gel em presença de dodecil sulfato de sódio. A enzima tem um ponto isoelétrico de 5,2 por eletroforese em gel de poliacrilamida contendo anfoline. Utilizando-se a reaçäo de intercâmbio ATP-PPi foram determinadas as condiçöes ótimas de ensaio e os valores aparentes de Km. através da mesma reaçäo foram também observados efeitos de cátions divalentes e diaminas em substituiçäo ao Mg2+ e efeitos de reagentes sulfidrílicos

Anti-threonyl-tRNA synthetase, a second myositis-related autoantibody.

An autoantibody known as PL-7 was found in the serum of four patients with myositis and one with a systemic lupus erythematosus-like syndrome. The PL-7 antigen is an 80,000 dalton polypeptide that coprecipitates with transfer RNA. In aminoacylation reactions, PL-7 IgG inhibited the charging of tRNA with threonine but had little or no effect on charging with other amino acids. Experimental antibodies raised against purified threonyl-tRNA synthetase recognized the same 80,000 dalton polypeptide, but tRNA was not coprecipitated. We conclude that PL-7 antibody is directed at threonyl-tRNA synthetase, and that different antigenic sites are recognized by the human and experimental autoantibodies. Our findings emphasize the link between myositis and autoimmunity to tRNA-related structures.

Purification and subunit structure studies of human placental threonyl-tRNA synthetase.

Human threonyl-tRNA synthetase has been purified from full-term placenta over 5000-fold with a 13% overall yield by a combination of affinity chromatographic methods and conventional procedures. The product was apparently homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the esterification of approximately 3500 nmol L-threonine to tRNA per min per mg of protein at 37 degrees, corresponding to a molecular activity of approximately 700/min. The apparent molecular weight of the native enzyme ranged from 210000 to 220000 by gel-filtration on Sephadex G-200, and was either approximately 110000 or 228000 by sucrose gradient centrifugation for different preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 85000 or 115000 for different preparations. These results suggest that human placental threonyl-tRNA synthetase has a subunit structure of the type alpha 2 with a subunit molecular weight of about 100000. However, some other molecular forms with lower specific activity were also found to exist under certain conditions.

Purification and structural characterization of rat liver threonyl transfer ribonucleic acid synthetase.

Threonyl-tRNA synthetase was purified approximately 500-fold from a high-speed supernatant fraction of rat liver. The purified enzyme was estimated to be > 95% pure from acrylamide gel electrophoresis under denaturing and nondenaturing conditions. Based on a native molecular weight from sedimentation equilibrium of 154000 and a subunit molecular weight of 85000 obtained bo sodium dodecyl sulfate gel electrophoresis, the protein appears to be an alpha 2 dimer. The alpha 2 structure was also supported by cross-linking studies of the native enzyme. The purified protein has an S20,w of 7.2 and an isoelectric point of 6.4. Amino acid analyses revealed no unusual features, but attempts at automated sequence analyses suggested that both amino termini are blocked. Preliminary carbohydrate analyses suggested that the enzyme is a glycoprotein. Antibodies were raised against the purified protein which could inactivate and precipitate threonyl-tRNA synthetase.

Rapid purification of threonyl-tRNA synthetase from Saccharomyces carlsbergensis by affinity elution from phosphocellulose.

Threonyl-tRNA synthetase [EC 6.1.1.3] of Saccharomyces carlsbergensis was highly purified by a simple enzyme-substrate affinity method. After calcium-gel treatment of the crude enzyme extract, ammonium sulfate precipitation, and removal of polynucleotides with DEAE-cellulose, the enzyme was nonspecifically adsorbed onto phosphocellulose (P-cellulose), and then eluted specifically with a linear concentration gradient of threonine tRNA from torula yeast (Candida (Torulopsis) utilis). The enzyme was purified 303-fold from the calcium-gel supernatant. This purification method is very simple and time-saving. The purified enzyme gave a single band on SDS-gel electrophoresis, indicating a molecular weight of 82,000, and exhibited a molecular weight of about 150,000 by gel filtration. This suggests that the enzyme consists of two identical subunits. Some kinetic properties of the pure enzyme are described.