Oxidative stress-responsive intracellular regulation specific for the angiostatic form of human tryptophanyl-tRNA synthetase

Tryptophanyl-tRNA synthetase (TrpRS) exists in two forms in human cells, i.e., a major form which represents the full-length protein and a truncated form (mini TrpRS) in which an NH(2)-terminal extension is deleted because of alternative splicing of its pre-mRNA. Mini TrpRS can act as an angiostatic factor, while full-length TrpRS is inactive. We herein show that an oxidized form of human glyceraldehyde-3-phosphate dehydrogenase (GapDH) interacts with both full-length and mini TrpRSs and specifically stimulates the aminoacylation potential of mini, but not full-length, TrpRS. In contrast, reduced GapDH did not bind to TrpRSs and did not influence their aminoacylation activity. Mutagenesis experiments clarified that the NH(2)-terminal Rossmann fold region of GapDH is crucial for its interaction with mini TrpRS as well as tRNA and for the regulation of its aminoacylation potential and suggested that monomeric GapDH can bind to mini TrpRS and stimulate its aminoacylation activity. These results suggest that the angiostatic human mini, but not the full-length, TrpRS may play an important role in the intracellular regulation of protein synthesis under conditions of oxidative stress.     

A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding

The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.     

Identification and characterization of human mitochondrial tryptophanyl-tRNA synthetase

A full-length cDNA clone encoding the human mitochondrial tryptophanyl-tRNA synthetase (h(mt)TrpRS) has been identified. The deduced amino acid sequence shows high homology to both the mitochondrial tryptophanyl-tRNA synthetase ((mt)TrpRS) from Saccharomyces cerevisiae and to different eubacterial forms of tryptophanyl-tRNA synthetase (TrpRS). Using the baculovirus expression system, we have expressed and purified the protein with a carboxyl-terminal histidine tag. The purified His-tagged h(mt)TrpRS catalyzes Trp-dependent exchange of PP(i) in the PP(i)-ATP exchange assay. Expression of h(mt)TrpRS in both human and insect cells leads to high levels of h(mt)TrpRS localizing to the mitochondria, and in insect cells the first 18 amino acids constitute the mitochondrial localization signal sequence. Until now the human cytoplasmic tryptophanyl-tRNA synthetase (hTrpRS) was thought to function as the h(mt)TrpRS, possibly in the form of a splice variant. However, no mitochondrial localization signal sequence was ever detected and the present identification of a different (mt)TrpRS almost certainly rules out that possibility. The h(mt)TrpRS shows kinetic properties similar to human mitochondrial phenylalanyl-tRNA synthetase (h(mt)PheRS), and h(mt)TrpRS is not induced by interferon-gamma as is hTrpRS.     

Autoadenylation of tryptophanyl-tRNA synthetase

Incubation of bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2) deprived of endogenous tryptophan, with [14C]ATP and without [gamma-32P]ATP, causes an appearance of radioactivity in protein due to formation of adenylated enzyme, [14C]AMP-E. Examination of the properties of the [14C]AMP-E thus obtained led to the conclusion that AMP is linked to the protein molecule via a macroergetic phosphoanhydride bond. ATP is formed when AMP-E is incubated with PPi. However, no tryptophanyl adenylate formation was observed when AMP-E was treated with tryptophan. The functional role of AMP-E remains obscure.     

Interferon induces tryptophanyl-tRNA synthetase expression in human fibroblasts.

A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.     

Phorbol ester stimulates expression of the human tryptophanyl-tRNA synthetase gene

The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on expression of the human interferon (IFN)-inducible tryptophanyl-tRNA synthetase (WRS) gene was studied. PMA caused an increase in the basal and IFN-induced WRS protein content in HeLa and HEK293 cultured cells. Besides, PMA upregulated WRS mRNA level in HeLa cells. Since PMA is known as a selective activator of protein kinase C (PKC) and is widely used to study the PKC-related pathways, these results show possible PKC involvement in regulation of the WRS gene expression. PKC inhibition by staurosporine (10 and 100 nM) had no effect on either basal or IFN-induced expression of WRS in either cell line. Consequently, PKC is not an indispensable element in WRS induction by IFN. Rather, PKC may activate WRS gene expression only by a distinct pathway.     

High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.

Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.     

Human tryptophanyl-tRNA synthetase binds with heme to enhance its aminoacylation activity

Mammalian tryptophanyl-tRNA synthetases (TrpRSs) are Zn2+-binding proteins that catalyze the aminoacylation of tRNATrp. The cellular expression level of human TrpRS is highly upregulated by interferon-gamma (IFN-gamma). In this study, a heme biosynthesis inhibitor, succinylacetone (SA), was found to inhibit cellular TrpRS activity in IFN-gamma-activated cells without affecting TrpRS protein expression. In addition, supplementation of lysates from the SA-treated cells with hemin fully restored TrpRS activity to control levels. Biochemical analyses using purified TrpRS demonstrated that heme can interact strongly with Zn2+-depleted human full-length TrpRS with a stoichiometric heme:protein ratio of 1:1 to enhance the aminoacylation activity significantly. In contrast, the Zn2+-bound form of TrpRS did not bind heme. Further studies using site-directed mutagenesis clarified that the Zn2+-unbound human H130R mutant cannot bind heme. These results provide the first evidence of the involvement of heme in regulation of TrpRS aminoacylation activity. The regulation mechanism and its physiological roles are discussed.     

Cloning and nucleotide sequence of the structural gene encoding for human tryptophanyl-tRNA synthetase.

A structural gene encoding bovine (b) tryptophanyl-tRNA synthetase (WRS) has recently been cloned and sequenced [Garret et al., Biochemistry 30 (1991) 7809-7817]. Using part of this sequence as a hybridisation probe we have cloned and sequenced a structural gene encoding human polypeptide highly homologous with two mammalian proteins, bWRS [Garret et al., Biochemistry 30 (1991) 7809-7817; EMBL accession No. X52113] and rabbit peptide chain release factor [Lee et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3508-3512]. Identification of the sequence encoding a human WRS is based on (i) the presence of 'HIGH' and 'KMSKS' structural motifs typical for class-I aminoacyl-tRNA synthetases [Eriani et al., Nature 347 (1990) 203-206]; (ii) coincidence of the number of SH groups per subunit estimated experimentally [Muench et al., Science 187 (1975) 1089-1091] and deduced from the cDNA sequence (six in both cases); (iii) close resemblance of two WRS polypeptides sequenced earlier [Muench et al., Science 187 (1975) 1089-1091] and the predicted structure in two different regions.     

Crystal structure of Pyrococcus horikoshii tryptophanyl-tRNA synthetase and structure-based phylogenetic analysis suggest an archaeal origin of tryptophanyl-tRNA synthetase

The ancient and ubiquitous aminoacyl-tRNA synthetases constitute a valuable model system for studying early evolutionary events. So far, the evolutionary relationship of tryptophanyl- and tyrosyl-tRNA synthetase (TrpRS and TyrRS) remains controversial. As TrpRS and TyrRS share low sequence homology but high structural similarity, a structure-based method would be advantageous for phylogenetic analysis of the enzymes. Here, we present the first crystal structure of an archaeal TrpRS, the structure of Pyrococcus horikoshii TrpRS (pTrpRS) in complex with tryptophanyl-5′ AMP (TrpAMP) at 3.0 Å resolution which demonstrates more similarities to its eukaryotic counterparts. With the pTrpRS structure, we perform a more complete structure-based phylogenetic study of TrpRS and TyrRS, which for the first time includes representatives from all three domains of life. Individually, each enzyme shows a similar evolutionary profile as observed in the sequence-based phylogenetic studies. However, TyrRSs from Archaea/Eucarya cluster with TrpRSs rather than their bacterial counterparts, and the root of TrpRS locates in the archaeal branch of TyrRS, indicating the archaeal origin of TrpRS. Moreover, the short distance between TrpRS and archaeal TyrRS and that between bacterial and archaeal TrpRS, together with the wide distribution of TrpRS, suggest that the emergence of TrpRS and subsequent acquisition by Bacteria occurred at early stages of evolution.     

Limited proteolysis of the tryptophanyl-tRNA synthetase.

Earlier studies have shown that native tryptophanyl-tRNA synthetase from beef pancreas is composed of two apparently identical subunits having a molecular weight of 60000 plus or minus 2000 each. Incubation of the pruified enzyme with trypsin under restrictive conditions results in splitting of each subunit to form an enzymatically inactive polypeptide chain of mol. wt 24500 plus or minus 1500. During proteolysis, two distinct intermediate forms of mol. wt 51000 plus or minus 2000 and 40000 plus or minus 2000 and fragments of mol. wt 14000 plus or minus 2500 are formed. The presence of substrates, viz. ATP, tryptophan or tryptophanyl adenylate, decreases the rate of proteolysis. However, a band pattern monitored by acrylamide gel electrophoresis is qualitatively indistinguishable from that obtained in the absence of substrates. Native and trypsin-modified subunits (the latter having a molecular weight of 24500) have been maleylated, reduced, carbosymethylated and subjected to exhaustive digestion by trypsin followed by peptide mapping. Comparison of the finger prints has shown that the trypsin-modified subunit represents a polypeptide with lowered content of dicarboxylic amino acids. That the number of peptides revealed after complete proteolysis of native and trypsin-modified subunits does not favour the presence of long repetitive sequences in each subunit, is at variance with some bacterial aminoacyl-tRNA synthetases. Study of the fluorescence polarisation of 1-anilino-8-napthalene sulphonate adsorbed on the dimeric tryptophanyl-tRNA synthetase, indicates that the molecule behaves as a complete entity in Brownian rotation. The trypsin-resistant end products, composed of two types of polypeptides (mol. wts 24500 and 14000), remain associated with each other. From the mol. wt of this associate it follows that each fragment is present in the associate in duplicate. When the purification procedure was carried out in the absence of a protease inhibitor, the active modified enzyme form was obtained. As judged from the molecular weight values, it is composed of two equal subunits corresponding to one of the products of limited proteolysis. The data presented are compatible with compact three-dimensional structure of tryptophanyl-tRNA synthetase having very limited regions exposed to exogenous or endogenous proteolysis.     

An interferon-induced protein with release factor activity is a tryptophanyl-tRNA synthetase.

Interferon gamma induces expression of a protein termed IFP 53 according to its molecular weight of 53 kDa. IFP 53 shows significant sequence homology to rabbit peptide chain release factor as well as to bovine tryptophanyl-tRNA synthetase. IFP 53 has been shown to possess release factor activity for the UGA stop codon. We demonstrate here, by using a recombinant IFP 53 fusion protein, that IFP 53 tryptophanylates tRNA. These data indicate that IFP 53 is a protein with two activities: peptide chain termination and aminoacylation.     

An insertion in loop L7 of human eosinophil-derived neurotoxin is crucial for its antiviral activity

The human eosinophil granule ribonuclease, eosinophil‐derived neurotoxin (EDN) has been shown to have antiviral activity against respiratory syncytial virus‐B (RSV‐B). Other closely related and more active RNases such as RNase A, onconase, and RNase k6 do not have any antiviral activity. A remarkable unique feature of EDN is a nine‐residue insertion in its carboxy‐terminal loop, L7 which is not present in RNase A, and differs in sequence from the corresponding loop in another eosinophil RNase, eosinophil cationic protein (ECP). ECP has a much lower antiviral activity as compared to EDN. The current study probed the role of loop L7 of EDN in its antiviral activity. Three residues in loop L7, Arg117, Pro120, and Gln122, which diverge between EDN, ECP, and RNase A, were mutated to alanine alone and in combination to generate single, double, and triple mutants. These mutants, despite having RNase activity had decreased antiviral activity towards RSV suggesting the involvement of loop L7 in the interaction of EDN with RSV. It appears that the mutations in loop L7 disrupt the interaction of protein with the viral capsid, thereby inhibiting its entry into the virions. The study demonstrates that besides the RNase activity, loop L7 is another important determinant for the antiviral activity of EDN. J. Cell. Biochem. 113: 3104–3112, 2012. © 2012 Wiley Periodicals, Inc.     

MSW, a yeast gene coding for mitochondrial tryptophanyl-tRNA synthetase

E569 and E606 are noncomplementing pet mutants of Saccharomyces cerevisiae. Both strains are defective in mitochondrial protein synthesis and as a result exhibit a pleiotropic deficiency in respiratory components that are translated on mitochondrial ribosomes. The wild type gene MSW capable of complementing the protein synthesis defect has been cloned by transformation of one of the mutants with a genomic library of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein with a molecular weight of 42,414 which is 37 and 39% identical to the tryptophanyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus, respectively. A strain containing an insertion in the chromosomal copy of MSW was constructed by in situ gene replacement. This mutant fails to charge mitochondrial tryptophanyl-tRNA providing further evidence that MSW is the structural gene for mitochondrial tryptophanyl tRNA synthetase. The existence of another gene coding for the cytoplasmic tryptophanyl-tRNA synthetase is inferred from the observation that mutations in MSW are not lethal but only result in a respiratory deficiency.     

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).