Eukaryotic Elongation Factor eEF1A Research Articles

Functional Analysis of Wheat NAC Transcription Factor, TaNAC069, in Regulating Resistance of Wheat to Leaf Rust Fungus.

NAC transcription factors are one of the largest transcription factor families having functions in a variety of stress responses. Few NACs have been reported for interactions between wheat and the wheat rust fungus Puccinia triticina (Pt). In this study, based on analysis of RNA-seq data from wheat line TcLr19 inoculated by Pt, the NAC transcription factor TaNAC069 was cloned from wheat, and its transcriptional activity and homologous dimer formation were verified. Quantitative real-time PCR analysis showed that the expression of TaNAC069 was induced by Pt and associated signaling molecules. To further characterize the function of the TaNAC069 gene in wheat resistance to Pt, virus-induced gene silencing (VIGS) was utilized, and it revealed that Pt resistance in TaNAC069-silenced plants was significantly reduced. Potential interaction targets of TaNAC069 from wheat and Pt were screened and identified by yeast two-hybrid technology. Eukaryotic elongation factor eEF1A, CBSX3 protein, and cold acclimation protein WCOR410c were screened by yeast one-hybrid technology. The results indicate that the TaNAC069 gene plays a positive regulatory role in wheat resistance to Pt, laying a good foundation to analyze the molecular mechanisms of TaNAC069 and its functional role in wheat resistance to Pt.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon–anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the ‘GTP’- and ‘GDP’-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

Abstract 5467: Role of the eukaryotic elongation factor eEF1A in the mechanism of action of Aplidin

Abstract Aplidin is a cyclic depsipeptide of the family of didemnins, originally isolated from the colonial tunicate Aplidium albicans. Aplidin is being evaluated in a phase III clinical trial in patients with relapse or refractory multiple myeloma. As part of its antitumor activity, Aplidin induces rapid oxidative stress, activation of Rac1 and phosphorylation of p38 and JNK1 stress kinases, which together trigger the apoptotic death of tumor cells. Didemnin B (DB), a molecule closely related to Aplidin, has been previously shown to interact with the GTP-bound conformation of the eukaryotic elongation factor eEF1A, an interaction that was related to the didemnin's B ability to inhibit protein synthesis (J. Biol. Chem. 1994, 269:15411-14). A structural model for this interaction has been proposed (J. Med. Chem. 2004, 47:4439-52). We wanted to investigate whether eEF1A had any role in the mechanism of action of Aplidin. Using the DARTS technique, we observed that Aplidin treatment of tumor cells and subsequent digestion of the cellular extracts with different proteases, resulted in a significant increase in the stabililty of eEF1A against protease digestion, suggesting a direct effect of Aplidin on this protein. We previously generated, by continuous exposure to increasing concentrations of the drug, a HeLa derivative cell line (HeLa-APL-R) that showed specific resistance to Aplidin as well as to other related didemnins and tamandarins (Br. J. Cancer 2004, 91:1405-13). We investigated whether there was any difference in the expression levels of eEF1A between HeLa-wt and HeLa-APL-R cell lines. Since two isoforms of the elongation factor are expressed in tumor cells, eEF1A1 and eEF1A2, we checked the relative amount of both proteins at the mRNA and protein levels using DNA microarrays and iTRAQ, respectively. Remarkably, we observed that the mRNA and protein levels of eEF1A2 isoform were lower in HeLa-APL-R resistant cells as compared to their parental cell line. No significant changes were seen in the levels of eEF1A1. The reduced levels of eEF1A2 protein in HeLa-APL-R cells were further confirmed by western blotting using isoform-specific antibodies. To explore the effect of the restoration of the eEF1A2 levels in the HeLa resistant clone, we generated two cell lines stably overexpressing eEF1A1 or eEF1A2 and checked their sensitivity to Aplidin in dose-response cytotoxicity experiments. Both cell lines partially recovered the sensitivity to Aplidin, with the eEF1A2-overexpressing cell line having an even slightly higher sensitivity to the compound. In eEF1A overexpressing cells, Aplidin induced a robust cytostatic effect. At the molecular level, Aplidin induced the phosphorylation of p38 as well as ERK MAPKs, but not JNK phosphorylation or PARP cleavage, two key events in the cytotoxic signaling of the drug. These results could indicate a role of eEF1A in the biological activity of Aplidin in tumor cells. Citation Format: Alejandro Losada, Juan F. Martínez-Leal, Federico Gago, Carmen Cuevas, Luis F. Garcia-Fernández, Carlos M. Galmarini. Role of the eukaryotic elongation factor eEF1A in the mechanism of action of Aplidin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2014-5467

Chapter 19 - Strategies for Investigating Nuclear–Cytoplasmic tRNA Dynamics in Yeast and Mammalian Cells

Nuclear-cytoplasmic tRNA transport involves multiple pathways that are segregated by the involvement of distinct proteins. The tRNA export process begins in the nucleolus, where the functionality of newly produced tRNAs are tested by aminoacylation, and ends with the delivery of the exported aminoacyl tRNAs to the eukaryotic elongation factor eEF-1A for utilization in protein synthesis in the cytoplasm. Recent studies have identified a number of proteins that participate in nuclear tRNA export in both yeast and mammals. However, genetic and biochemical evidence suggest that additional components, which have yet to be identified, also participate in nuclear-cytoplasmic tRNA trafficking. Here we review key strategies that have led to the identification and characterization of proteins that are involved in the nuclear tRNA export process in yeasts and mammals. The approaches described will greatly facilitate the identification and delineation of the roles of new proteins involved in nuclear export of tRNAs to the cytoplasm.

Effector Glycosyltransferases in Legionella

Legionella causes severe pneumonia in humans. The pathogen produces an array of effectors, which interfere with host cell functions. Among them are the glucosyltransferases Lgt1, Lgt2 and Lgt3 from L. pneumophila. Lgt1 and Lgt2 are produced predominately in the post-exponential phase of bacterial growth, while synthesis of Lgt3 is induced mainly in the lag-phase before intracellular replication of bacteria starts. Lgt glucosyltransferases are structurally similar to clostridial glucosylating toxins. The enzymes use UDP–glucose as a donor substrate and modify eukaryotic elongation factor eEF1A at serine-53. This modification results in inhibition of protein synthesis and death of target cells.In addition to Lgts, Legionella genomes disclose several genes, coding for effector proteins likely to possess glycosyltransferase activities, including SetA (subversion of eukaryotic vesicle trafficking A), which influences vesicular trafficking in the yeast model system and displays tropism for late endosomal/lysosomal compartments of mammalian cells. This review mainly discusses recent results on the structure–function relationship of Lgt glucosyltransferases.

Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.

Cex1p is a novel cytoplasmic component of the Saccharomyces cerevisiae nuclear tRNA export machinery

The Saccharomyces cerevisiae Yor112wp, which we named Cex1p, was identified using a yeast tRNA three-hybrid interaction approach and an in vivo nuclear tRNA export assay as a cytoplasmic component of the nuclear tRNA export machinery. Cex1p binds tRNA saturably, and associates with the nuclear pore complex by interacting directly with Nup116p. Cex1p co-purifies with the nuclear tRNA export receptors Los1p and Msn5p, the eukaryotic elongation factor eEF-1A, which delivers aminoacylated tRNAs to the ribosome, and the RanGTPase Gsp1p, but not with Cca1p, a tRNA maturation enzyme that facilitates translocation of non-aminoacylated tRNAs across the nuclear pore complex. Depletion of Cex1p and eEF-1A or Los1p significantly reduced the efficiency of nuclear tRNA export. Cex1p interacts with Los1p but not with eEF-1A in vitro. These findings suggest that Cex1p is a component of the nuclear aminoacylation-dependent tRNA export pathway in S. cerevisiae. They also suggest that Cex1p collects aminoacyl-tRNAs from the nuclear export receptors at the cytoplasmic side of the nuclear pore complex, and transfers them to eEF-1A using a channelling mechanism.

The product of the mammalian orthologue of the Saccharomyces cerevisiaeHBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity

We describe here the cloning and sequencing of human and mouse cDNAs encoding a putative GTP binding protein. Sequence comparison shows that these cDNAs (named eRFS) are likely to represent the orthologues of the yeast Saccharomyces cerevisiae HBS1 gene and that the C-terminal domains of the encoded proteins share structural features with eukaryotic elongation factor eEF-1A and release factor 3 (eRF3) families. The phylogenetic analysis suggests that eRFS proteins and Hbs1p form a cluster of orthologous sequences branching with the eRF3 family. Nevertheless, in yeast, the human eRFS protein and Hbs1p do not complement eRF3/Sup35p thermosensitive mutation and do not interact with eRF1.