Functional Interaction of Mammalian Valyl-tRNA Synthetase with Elongation Factor EF-1α in the Complex with EF-1H

Negrutskii, B. S.; Shalak, V. F.; Kerjan, Pierre; El'skaya, A. V.; Mirande, Marc

doi:10.1074/jbc.274.8.4545

Cited by 80 publications

(75 citation statements)

References 33 publications

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“…For example, the N-terminal extension of rat aspartyl-tRNA synthetase facilitates the release of aminoacylated tRNA to elongation factor (22,23), and the aminoacylation reaction of rabbit valyl-tRNA synthetase is enhanced by interaction with elongation factor EF-1H (24). The N-terminal extension of yeast glutaminyl-tRNA synthetase promotes specific recognition of its cognate tRNA (25), and the C-terminal appendix of E. coli methionyl-tRNA synthetase helps to dock its cognate tRNA to the active site (26).…”

Section: Discussionmentioning

confidence: 99%

Precursor of Pro-apoptotic Cytokine Modulates Aminoacylation Activity of tRNA Synthetase

Park¹,

Jung²,

Lee³

et al. 1999

Journal of Biological Chemistry

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Endothelial monocyte activating polypeptide II (EMA-PII) is a cytokine that is specifically induced by apoptosis.Its precursor (pro-EMAPII) has been suggested to be identical to p43, which is associated with the multi-tRNA synthetase complex. Herein, we have demonstrated that the N-terminal domain of pro-EMAPII interacts with the Nterminal extension of human cytoplasmic arginyl-tRNA synthetase (RRS) using genetic and immunoprecipitation analyses. Aminoacylation activity of RRS was enhanced about 2.5-fold by the interaction with pro-EMAPII but not with its N-or C-terminal domains alone. The N-terminal extension of RRS was not required for enzyme activity but did mediate activity stimulation by pro-EMAPII. Pro-EMAPII reduced the apparent K m of RRS to tRNA, whereas the k cat value remained unchanged. Therefore, the precursor of EMAPII is a multi-functional protein that assists aminoacylation in normal cells and releases the functional cytokine upon apoptosis.Aminoacyl-tRNA synthetases (ARSs) 1 catalyze ligation of their cognate amino acids to specific tRNAs. Although basic architecture of the core domain is well conserved among ARSs, unique peptide extensions have been found in the N-or Cterminal ends of metazoan enzymes (1-3). Although these extensions have been thought to be involved in heterologous molecular interactions, their functional significance is not yet understood.A macromolecular protein complex consisting of at least nine different ARSs has been found in higher eukaryotes (1-3). This multi-ARS complex also contains three nonsynthetase components, p18, p38, and p43 whose functions are not clear (4 -7). Among these nonsynthetase components, p43 has been proposed to be a precursor of a tumor-specific cytokine, endothelial monocyte-activating polypeptide II (EMAPII) based on over 80% sequence identity between the two proteins (6). EMAPII was originally identified in the culture medium of murine fibrosarcoma cells induced by methylcholanthrene A (8). It triggers an acute inflammatory response (9, 10) and is involved in development-related apoptosis (11).The precursor for EMAPII (pro-EMAPII) is processed at the Asp residue of ASTD/S sequence to release the C-terminal cytokine domain of 23 kDa (11). Its C-terminal domain shares homology with the C-terminal parts of methionyl-tRNA synthetases of prokaryotes, archaea and nematode, and also a yeast protein, Arc1p/G4p, which interacts with methionyl-and glutamyl-tRNA synthetases. The N-terminal domain of pro-EMAPII does not show homology to any known proteins, and its function has not been understood.EMAPII is expressed in a wide range of cell lines and normal tissues (12) and its mRNA level is unchanged during apoptosis (11) although its production is induced by apoptosis. The present work was designed to address whether pro-EMAPII is identical to p43 and to understand its function in the normal cell. The results showed that pro-EMAPII is associated with the N-terminal extension of human arginyl-tRNA synthetase (RRS), facilitating aminoacylation reaction. EXPE...

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Section: Discussionmentioning

confidence: 99%

Precursor of Pro-apoptotic Cytokine Modulates Aminoacylation Activity of tRNA Synthetase

Park¹,

Jung²,

Lee³

et al. 1999

Journal of Biological Chemistry

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“…To obtain the GTP form of bacterial EF1A, the factor was incubated with 100 lM GTP in the incubation mixture containing 25 mM Tris/HCl, pH 7.5, 50 mM NH 4 Cl, 10 mM MgCl 2 , 1 mM dithiothreitol, 0.5 mM EDTA in the presence of 30 lgAEmL )1 phosphoenolpyruvate kinase and 2 mM phosphoenolpyruvate to remove traces of GDP. Incubation was carried out at 30°C for 15 min, and the EF1AAEGTP preparation was used immediately.…”

Section: Preparation Of Bacterial Ef1aaegtpmentioning

confidence: 99%

“…There is an increasing body of evidence for special structural organization of the protein synthesis machinery in the higher eukaryotic cells. The existence of multimolecular complexes of ARS [1], initiation factors [2] and eEF1 [3,4], ribosome-ARS interactions [5][6][7], and the association of translation components with cytoskeletal framework [8] are among the important signs of the protein synthesis compartmentalization. Moreover, detailed fluorescence-based measurements of translation in living dendrites have visualized the mammalian protein synthesis compartments in situ [9].…”

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confidence: 99%

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Petrushenko

Budkevich

Shalak

et al. 2002

European Journal of Biochemistry

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Multimolecular complexes involving the eukaryotic elongation factor 1A (eEF1A) have been suggested to play an important role in the channeling (vectorial transfer) of tRNA during protein synthesis [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47–78]. Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl‐tRNA, the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich, T.V., Shalak, V.F. & El'skaya, A.V. (1997) FEBS Lett. 407, 13–17]. The [eEF1A·GDP·tRNA] complex has been hypothesized to interact with aminoacyl‐tRNA synthetase (ARS) resulting in a quaternary complex where uncharged tRNA is transferred to the enzyme for aminoacylation. Here we present the data on association of the [eEF1A·GDP·tRNA] complex with phenylalanyl‐tRNA synthetase (PheRS), e.g. the formation of the above quaternary complex detected by the gel‐retardation and surface plasmon resonance techniques. To estimate the stability of the novel ternary and quaternary complexes of eEF1A the fluorescence method and BIAcore analysis were used. The dissociation constants for the [eEF1A·GDP·tRNA] and [eEF1A·GDP·tRNAPhe·PheRS] complexes were found to be 20 nm and 9 nm, respectively. We also revealed a direct interaction of PheRS with eEF1A in the absence of tRNAPhe (Kd = 21 nm). However, the addition of tRNAPhe accelerated eEF1A·GDP binding to the enzyme. A possible role of these stable novel ternary and quaternary complexes of eEF1A·GDP with tRNA and ARS in the channeled elongation cycle is discussed.

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“…Interestingly, the eEF1B␤ subunit is glutathionylated in response to oxidative stress (58) and the eEF1B␥ subunit contains a redox-active pair of cysteine residues (59). These post-translational modifications might affect protein synthesis either by direct alterations in the nucleotide-exchange activity of eEF1B, or through the allosteric effects that interactions with this complex have on tRNA synthetases (60,61).…”

Section: Resultsmentioning

confidence: 99%

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

Vickers

Wyllie

Fairlamb

2004

Journal of Biological Chemistry

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In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified ␣, ␤, and ␥ subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg ؊1 ). Kinetic studies suggest a ternary complex mechanism, with K m values of 140 M for trypanothione and 7.4 mM for cumene hydroperoxide and k cat ‫؍‬ 25 s ؊1 . Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.Parasitic protozoa of the family trypanosomatidae cause disease and death throughout the tropical and subtropical world. Trypanosoma brucei infections are estimated to cause ϳ400,000 cases of sleeping sickness per year; Trypanosoma cruzi, the cause of Chagas' disease, chronically infects ϳ17 million people and Leishmania spp. are thought to cause 2 million cases of leishmaniasis per year. 1 Current chemotherapies for these diseases are, on the whole, ineffective and toxic (2, 3), whereas effective vaccines may never be developed. The development of effective treatments for these infections is therefore an urgent necessity.New anti-parasitic drugs can be developed from inhibitors of biochemical pathways that are essential for parasite survival but absent from the host. One such target is the thiol metabolism of trypanosomatids. Uniquely, this is dependent upon trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine or T[SH] 2 ) 2 (4), whereas their human hosts use glutathione ((␥-L-glutamyl-L-cysteinylglycine or GSH). Trypanothione is involved in protective processes, defending against oxidative stress through peroxidase systems (5), against reactive aldehydes through the glyoxalase system (6, 7), and against toxic xenobiotics through the trypanothione S-transferase (TST) (8). All these processes depend upon trypanothione being maintained in its dithiol form by trypanothione reductase. This NADPH-dependent flavoenzyme is essential to the trypanosomatids (9, 10) as, in these organisms, it is the only known route for the transfer of reducing equivalents from NADPH to low molecular mass thiols. In addition, because these parasites lack an...

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Functional Interaction of Mammalian Valyl-tRNA Synthetase with Elongation Factor EF-1α in the Complex with EF-1H

Cited by 80 publications

References 33 publications

Precursor of Pro-apoptotic Cytokine Modulates Aminoacylation Activity of tRNA Synthetase

Precursor of Pro-apoptotic Cytokine Modulates Aminoacylation Activity of tRNA Synthetase

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

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