Mammalian aminoacyl-tRNA Synthetases Research Articles

High-Throughput Screening for Protein Synthesis Inhibitors Targeting Aminoacyl-tRNA Synthetases

Aminoacylation has been implicated in a wide variety of cancers. Aminoacyl-tRNA synthetases (ARSs) exist in large excess in tumor cells due to their increased demand for translation, whereas most other protein-synthesis apparatuses are quantitatively limited. Among other components that constitute the translation machinery—namely, tRNA, amino acid, ATP, and ARS—ARS is the only target that can be blocked by small molecules. No constitutively active ARSs have been reported, and mutations of ARS can cause inaccurate substrate recognition and malformation of the multi-ARS complex (MSC). Hence, interference of the activity is expected to be independent of genotype without developing resistance. Here, we report a high-throughput screening (HTS) system to find mammalian ARS inhibitors. The rabbit–reticulocyte lysate we used closely resembles both the individual and complexed structures of human ARSs, and it may predispose active compounds that are readily applicable for humankind. This assay was further validated because it identified familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for our purpose. The assay demonstrated excellent quality control (QC) parameters and reproducibility, and is proven ready for further HTS campaigns with large chemical libraries.

Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping

Over the past decade, the identification of cancer-associated factors has been a subject of primary interest not only for understanding the basic mechanisms of tumorigenesis but also for discovering the associated therapeutic targets. However, aminoacyl-tRNA synthetases (ARSs) have been overlooked, mostly because many assumed that they were simply 'housekeepers' that were involved in protein synthesis. Mammalian ARSs have evolved many additional domains that are not necessarily linked to their catalytic activities. With these domains, they interact with diverse regulatory factors. In addition, the expression of some ARSs is dynamically changed depending on various cellular types and stresses. This Analysis article addresses the potential pathophysiological implications of ARSs in tumorigenesis.

Not Just Because It Is There: Aminoacyl-tRNA Synthetases Gain Control of the Cell

In a recent issue of Molecular Cell, Jia et al. (2008) demonstrate that time-controlled repression of interferon-induced transcripts depends upon the interaction between an RNA structure in these transcripts and protein domains harbored by a mammalian aminoacyl-tRNA synthetase.

Occurrence of the aminoacyl-tRNA synthetases in high-molecular weight complexes correlates with the size of substrate amino acids

One of the distinctive and mysterious features of mammalian aminoacyl-tRNA synthetases (AARSs) is the existence of stable high-molecular weight complexes containing 10 out of 20 AARSs. The composition and structure of these complexes are conserved among multicellular animals. No specific function associated with these structures has been found, and there is no evident rationale for a particular separation of AARSs in “complex-bound” and “free” forms. We have demonstrated a strong association between the occurrence of AARSs in the complexes and the volume of their substrate amino acids. The significance of this association is discussed in terms of the structural organization of translation in the living cell.

The p38 subunit of the aminoacyl-tRNA synthetase complex is a Parkin substrate: linking protein biosynthesis and neurodegeneration.

Parkinson's disease (PD) is a severe neurological disorder, characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the presence of Lewy bodies (LBs). The discovery of genes responsible for familial forms of the disease has provided insights into its pathogenesis. Mutations in the parkin gene, which encodes an E3 ubiquitin-protein ligase involved in the ubiquitylation and proteasomal degradation of specific protein substrates, have been found in nearly 50% of patients with autosomal-recessive early-onset parkinsonism. The abnormal accumulation of substrates due to loss of Parkin function may be the cause of neurodegeneration in parkin-related parkinsonism. Here, we demonstrate that Parkin interacts with, ubiquitylates and promotes the degradation of p38, a key structural component of the mammalian aminoacyl-tRNA synthetase complex. We found that the ubiquitylation of p38 is abrogated by truncated variants of Parkin lacking essential functional domains, but not by the pathogenic Lys161Asn point mutant. Expression of p38 in COS7 cells resulted in the formation of aggresome-like inclusions in which Parkin was systematically sequestered. In the human dopaminergic neuroblastoma-derived SH-SY5Y cell line, Parkin promoted the formation of ubiquitylated p38-positive inclusions. Moreover, the overexpression of p38 in SH-SY5Y cells caused significant cell death against which Parkin provided protection. Analysis of p38 expression in the human adult midbrain revealed strong immunoreactivity in normal dopaminergic neurons and the labeling of LBs in idiopathic PD. This suggests that p38 plays a role in the pathogenesis of PD, opening the way for a detailed examination of its potential non-canonical role in neurodegeneration.

Molecular network and functional implications of macromolecular tRNA synthetase complex

Understanding the complex network and multi-functionality of proteins is one of the main objectives of post-genome research. Aminoacyl-tRNA synthetases (ARSs) are the family of enzymes that are essential for cellular protein synthesis and viability that catalyze the attachment of specific amino acids to their cognate tRNAs. However, a lot of evidence has shown that these enzymes are multi-functional proteins that are involved in diverse cellular processes, such as tRNA processing, RNA splicing and trafficking, rRNA synthesis, apoptosis, angiogenesis, and inflammation. In addition, mammalian ARSs form a macromolecular complex with three auxiliary factors or with the elongation factor complex. Although the functional meaning and physiological significance of these complexes are poorly understood, recent data on the molecular interactions among the components for the multi-ARS complex are beginning to provide insights into the structural organization and cellular functions. In this review, the molecular mechanism for the assembly and functional implications of the multi-ARS complex will be discussed.

Dose-dependent Biphasic Activity of tRNA Synthetase-associating Factor, p43, in Angiogenesis

Mammalian aminoacyl tRNA synthetases form a macromolecular protein complex with three non-enzymatic cofactors. Among these factors, p43 is also secreted to work as a cytokine on endothelial as well as immune cells. Here we investigated the activity of p43 in angiogenesis and determined the related mediators. It promoted the migration of endothelial cells at low dose but induced their apoptosis at high dose. p43 at low concentration activated extracellular signal-regulating kinase, which resulted in the induction and activation of matrix metalloproteinase 9. In contrast, p43 at high concentration activated Jun N-terminal kinase, which mediated apoptosis of endothelial cells. These results suggest that p43 is a novel cytokine playing a dose-dependent biphasic role in angiogenesis.

Novel regulatory interactions and activities of mammalian tRNA synthetases.

Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs, thereby ensuring the faithful translation of genetic code. In addition to their enzymatic function, these enzymes have been discovered to regulate various cellular functions such as tRNA export, ribosomal RNA synthesis, apoptosis, inflammation and angiogenesis in mammalian. The insights into the noncanonical activities of these enzymes have been obtained from their unique cellular localization, interacting partners, isoform generation and expression control. Mammalian ARSs also form a macromolecular protein complex with a few auxiliary factors. Although the physiological significance of this complex is poorly understood, it also supports the potential of mammalian ARSs as sophisticated multifunctional proteins for regulating various cellular procedures. In this review, the novel regulatory activities of mammalian ARSs will be discussed in different biological processes.

Interaction Network of Human Aminoacyl-tRNA Synthetases and Subunits of Elongation Factor 1 Complex

Aminoacyl-tRNA synthetases (ARSs) ligate amino acids to their cognate tRNAs. It has been suggested that mammalian ARSs are linked to the EF-1 complex for efficient channeling of aminoacyl tRNAs to ribosome. Here we systemically investigated possible interactions between human ARSs and the subunits of EF-1 (α, β, γ, and δ) using a yeast two-hybrid assay. Among the 80 tested pairs, leucyl- and histidyl-tRNA synthetases were found to make strong and specific interaction with the EF-1γ and β while glu-proly-, glutaminyl-, alanyl-, aspartyl-, lysyl-, phenylalanyl-, glycyl-, and tryptophanyl-tRNA synthetases showed moderate interactions with the different EF-1 subunits. The interactions of leucyl- and histidyl-tRNA synthetase with the EF-1 complex were confirmed by immunoprecipitation and in vitro pull-down experiments. Interestingly, the aminoacylation activities of these two enzymes, but not other ARSs, were stimulated by the cofactor of EF-1, GTP. These data suggest that a systematic interaction network may exist between mammalian ARSs and EF-1 subunits probably to enhance the efficiency of in vivo protein synthesis.

Import of amber and ochre suppressor tRNAs into mammalian cells: a general approach to site-specific insertion of amino acid analogues into proteins.

A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoacylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only requirement is that the suppressor tRNA must not be a substrate for any of the cellular aminoacyl-tRNA synthetases. Here, we describe conditions for the import of amber and ochre suppressor tRNAs derived from Escherichia coli initiator tRNA into mammalian COS1 cells, and we present evidence for their activity in the specific suppression of amber (UAG) and ochre (UAA) codons, respectively. We show that an aminoacylated amber suppressor tRNA (supF) derived from the E. coli tyrosine tRNA can be imported into COS1 cells and acts as a suppressor of amber codons, whereas the same suppressor tRNA imported without prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl-tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid analogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility further of importing a mixture of amber and ochre suppressor tRNAs for the insertion of two different amino acid analogues into a protein and the potential use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations.

Catalytic Peptide of Human Glutaminyl-tRNA Synthetase Is Essential for Its Assembly to the Aminoacyl-tRNA Synthetase Complex

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.

P70. Inhibition by bisphosphonates of mammalian aminoacyl-tRNA synthetase enzymes

P70. Inhibition by bisphosphonates of mammalian aminoacyl-tRNA synthetase enzymes

Interferon inducibility of mammalian tryptophanyl-tRNA synthetase: new perspectives

Mammalian aminoacyl-tRNA synthetases are indispensible components of the cell's protein-synthesizing machinery. Suprisingly, recent experiments have demonstrated that synthesis of tryptophanyl-tRNA synthetase (WRS) is markedly enhanced after incubation of human cells with interferon. Why is this housekeeping enzyme interferon-inducible? Several hypotheses have been suggested. One hypothesis, that premature termination of protein synthesis was involved, was boosted by the discovery that the deduced amino acid sequence of the mammalian peptide chain release factor (RF) closely resembled that of WRS. Further investigation, however, suggests that the DNA encoding RF was wrongly identified and in fact encodes a rabbit WRS subunit. Other hypotheses on the interferon-inducibility of WRS, including the possibility that the protein performs other, regulatory functions in addition to its core enzymic activity, remain to be explored.

Co-purification of the aminoacyl-tRNA synthetase complex with the elongation factor eEF1

A multi-enzyme complex of mammalian aminoacyl-tRNA synthetases was isolated from rabbit reticulocytes, and purified by polyethylene glycol fractionation and gel filtration on Biogel A15m and affinity chromatography on tRNA-Sepharose. The synthetase complex contains nine synthetase activities, and the corresponding proteins as analyzed by SDS polyacrylamide gel electrophoresis. Three of the proteins showed the identical subunit molecular weights to those of the reticulocyte's elongation factor eEF1H. The eEF1α protein could not be removed by second tRNA-Sepharose column chromatography, or gel filtration on Biogel A5m or Biogel A15m. Antibodies against eEF1α react with the purified synthetase complex on the basis of dot blot analysis. This finding should provide new clues for elucidating the structural organization of the mammalian protein biosynthetic machinery.

Glutaminyl-tRNA synthetase as a component of the high-molecular weight complex of human aminoacyl-tRNA synthetases. An immunological study

The human glutaminyl-tRNA synthetase is three times larger than the corresponding bacterial and twice as large as the yeast enzyme. It is possible that the additional sequences of the human glutaminyl-tRNA synthetase are required for the formation of the multienzyme complex which is known to include several of aminoacyl-tRNA synthetases in mammalian cells. To address this point we prepared antibodies against three regions of the human glutaminyl-tRNA synthetase, namely against its enzymatically important core region, and against two sections in its large C-terminal extension. In intact multienzyme complexes the core region was accessible to specific antibody binding. However, the C-terminal sections became available to specific antibody binding only when certain components of the multienzyme complex were either absent or degraded. These findings allow first conclusions as to the relative positon of some components in the mammalian aminoacyl-tRNA synthetase complex.

CDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase

Eight of the mammalian aminoacyl-tRNA synthetases associate as a multienzyme complex, whereas prokaryotic and low eukaryotic synthetases occur only as free soluble enzymes. Association of the synthetases may result in effective compartmentalization of synthetases and suggests the association of the entire protein biosynthetic machinery. To elucidate the structural elements and the nature of the molecular interactions involved in the association of the synthetases, we have cloned and sequenced the complementary DNA coding human aspartyl-tRNA synthetase. The full length cDNA encodes an open reading frame of 500 amino acids with 56% identity with yeast aspartyl-tRNA synthetase. The similarity with yeast aspartyl-tRNA synthetase is unevenly distributed with a high percent of identity at the C-terminus and relatively low identity at the N-terminus. The N-terminal sequence strongly prefers an alpha-helical secondary structure and shows amphiphilic characteristics. Further comparison with the yeast synthetases showed that the basic positively charged helixes in yeast synthetases are evolved to a neutral amphiphilic helix in this mammalian synthetase. The mammalian neutral amphiphilic helix is so far unique among all known sequences of bacterial, yeast, and mammalian synthetases and may account for the association of synthetases in the synthetase complex.

Structural organization of the multienzyme complex of mammalian aminoacyl-tRNA synthetases.

The multienzyme complexes of mammalian aminoacyl-tRNA synthetases were purified from rat liver, rabbit liver, and rabbit reticulocytes according to the procedure slightly modified from Kellermann et al. [Kellermann, O., Brevet, A., Tonetti, H., & Waller, J.-P. (1979) Eur. J. Biochem. 99, 541-550]. Three forms of the synthetase complex with slightly different protein compositions were identified, suggesting a microheterogeneity of the synthetase complex. The hydrodynamic properties and the protein composition of the purified complexes were determined. The electron micrographs of the complex showed mostly amorphous particles and some hollow rings with an outer diameter of 164 A and an inner diameter of 42 A. The predicted hydrodynamic properties of several models of the complex were calculated. The properties of a ring model appear to best fit with those of the synthetase complex.

Proteolytic signal sequences (PEST) in the mammalian aminoacyl-tRNA synthetase complex

Eight aminoacyl-tRNA synthetases together with three unidentified proteins are associated as a multi-enzyme complex in mammalian cells. Partial peptide sequences for lysyl- and aspartyl-tRNA synthetases are determined and no highly hydrophobic peptides are found. The partial amino acid sequences for two of the unidentified proteins in the complex are shown to have substantial homology and each has a number of unique sequences. The results suggest that the two unidentified proteins are fragments of synthetases. The partial sequences revealed the presence of PEST sequences in at least three proteins. Inasmuch as PEST sequences are signals for intracellular degradation, the mammalian synthetase complex may have evolved to protect these synthetases against intracellular proteolysis.

Generation of multiple forms of methionyl-tRNA synthetase from the multi-enzyme complex of mammalian aminoacyl-tRNA synthetases by endogenous proteolysis

Methionyl-tRNA synthetase occurs free and as high-molecular-weight multi-enzyme complexes in rat liver. The free form is purified to near homogeneity by conventional column chromatography and affinity chromatography on tRNA-Sepharose. The native molecular weight of free methionyl-tRNA synthetase is 64 500, based on its sedimentation coefficient of 4.5 S and Stokes radius of 33 Å. The free methionyl-tRNA synthetase apparently belongs to α-type subunit structure, since the subunit molecular weight is 68000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Methionyl-tRNA synthetase is dissociated from the high-molecular-weight synthetase complex by controlled trypsinization, according to Kellermann, O., Viel, C. and Waller, J.P. (Eur. J. Biochem. 88 (1978) 197–204). The dissociated, free methionyl-tRNA synthetase is subsequently purified to near homogeneity. The subunit structure of dissociated methionyl-tRNA synthetase is identical to that of endogenous free methionyl-tRNA synthetase. Anti-serum raised against M r 104000 protein in the synthetase complex, specifically inhibited methionyl-tRNA synthetase in both the free and the high-molecular-weight forms to the same extent. These results suggest that the occurrence of multiple forms of methionyl-tRNA synthetases in mammalian cells may, in part, be due to proteolytic cleavage.

Association of an aminoacyl-tRNA synthetase complex and of phenylalanyl-tRNA synthetase with the cytoskeletal framework fraction from mammalian cells

The intracellular distribution of several mammalian aminoacyl-tRNA synthetases was investigated by biochemical and immunocytological approaches. The fraction of aminoacyl-tRNA synthetases bound to the detergent-insoluble cytoskeletal framework obtained after extraction of NRK cells by 0.1 % Triton X-100 was estimated, by activity measurements, to about 80% for phenylalanyl-tRNA synthetase and 40% for the high-molecular-weight (HMW) complex containing the seven aminoacyl-tRNA synthetases specific for glutamic acid, isoleucine, leucine, methionine, glutamine, lysine, and arginine. This association was shown to be salt-dependent. The subcellular localization of these enzymes was examined using an immunocytoiogical approach. When cultured cells were fixed with paraformaldehyde and then permeabilized with Triton X-100, a fairly uniform cytoplasmic labelling was observed with antibodies directed to the aminoacyl-tRNA synthetase complex or to phenylalanyl-tRNA synthetase. By contrast, when cells were extracted with 0.1% Triton X-100 prior to fixation with paraformaldehyde, the staining patterns obtained with antibodies to aminoacyl-tRNA synthetases were very similar to that obtained with antibodies to rough endoplasmic reticulum, as assessed by single or double indirect immunofluorescence microscopy. These results suggest that free and bound forms of these aminoacyl-tRNA synthetases may coexist within the cell. In addition to cytoplasmic labelling, antibodies directed to phenylalanyl-tRNA synthetase stained the nucleus of rapidly growing cells. The possible significance of this finding is discussed.