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Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E-eIF4G interactions.

Yoffe Y, Léger M, Zinoviev A, Zuberek J, Darzynkiewicz E, Wagner G, Shapira M - Nucleic Acids Res. (2009)

Bottom Line: LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E.A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27).The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.

ABSTRACT
Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.

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LeishIF4G-3 interacts with LeishIF4E-1 and LeishIF4E-4 in vitro. GST or GST-LeishIF4G-3 fusion protein were immobilized on Glutathione-Agarose beads. The beads were incubated with supernatant of bacteria that express the different LeishIF4E isoforms or the mouse eIF4E ortholog (S). After extensive washes (W) the protein complexes were eluted (E), and analyzed by western blot with specific antibodies against the different eIF4Es.
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Figure 3: LeishIF4G-3 interacts with LeishIF4E-1 and LeishIF4E-4 in vitro. GST or GST-LeishIF4G-3 fusion protein were immobilized on Glutathione-Agarose beads. The beads were incubated with supernatant of bacteria that express the different LeishIF4E isoforms or the mouse eIF4E ortholog (S). After extensive washes (W) the protein complexes were eluted (E), and analyzed by western blot with specific antibodies against the different eIF4Es.

Mentions: A common feature of the cap-binding complex in eukaryotes is the interaction between eIF4E and eIF4G. Copurification over m7GTP-Sepharose column and comigration on sucrose gradients strongly suggest the ability of the parasite proteins to physically interact, although being indirect. Therefore, pull-down assays using a GST-LeishIF4G-3 fusion protein, or GST alone were expressed in bacteria and immobilized onto Glutathione-Agarose beads. These beads were incubated with the soluble extracts of bacteria, each expressing one of the four LeishIF4Es, or the mouse eIF4E ortholog. The complexes were eluted and their components were identified by western blot analysis using specific antibodies. As shown in Figure 3, LeishIF4G-3 pulled-down LeishIF4E-1 and LeishIF4E-4, but not LeishIF4E-2 or LeishIF4E-3, when compared with the GST control. No interaction was observed between LeishIF4G-3 and the mouse eIF4E, supporting the observed divergence between LeishIF4G-3 and its mammalian ortholog.Figure 3.


Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E-eIF4G interactions.

Yoffe Y, Léger M, Zinoviev A, Zuberek J, Darzynkiewicz E, Wagner G, Shapira M - Nucleic Acids Res. (2009)

LeishIF4G-3 interacts with LeishIF4E-1 and LeishIF4E-4 in vitro. GST or GST-LeishIF4G-3 fusion protein were immobilized on Glutathione-Agarose beads. The beads were incubated with supernatant of bacteria that express the different LeishIF4E isoforms or the mouse eIF4E ortholog (S). After extensive washes (W) the protein complexes were eluted (E), and analyzed by western blot with specific antibodies against the different eIF4Es.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691829&req=5

Figure 3: LeishIF4G-3 interacts with LeishIF4E-1 and LeishIF4E-4 in vitro. GST or GST-LeishIF4G-3 fusion protein were immobilized on Glutathione-Agarose beads. The beads were incubated with supernatant of bacteria that express the different LeishIF4E isoforms or the mouse eIF4E ortholog (S). After extensive washes (W) the protein complexes were eluted (E), and analyzed by western blot with specific antibodies against the different eIF4Es.
Mentions: A common feature of the cap-binding complex in eukaryotes is the interaction between eIF4E and eIF4G. Copurification over m7GTP-Sepharose column and comigration on sucrose gradients strongly suggest the ability of the parasite proteins to physically interact, although being indirect. Therefore, pull-down assays using a GST-LeishIF4G-3 fusion protein, or GST alone were expressed in bacteria and immobilized onto Glutathione-Agarose beads. These beads were incubated with the soluble extracts of bacteria, each expressing one of the four LeishIF4Es, or the mouse eIF4E ortholog. The complexes were eluted and their components were identified by western blot analysis using specific antibodies. As shown in Figure 3, LeishIF4G-3 pulled-down LeishIF4E-1 and LeishIF4E-4, but not LeishIF4E-2 or LeishIF4E-3, when compared with the GST control. No interaction was observed between LeishIF4G-3 and the mouse eIF4E, supporting the observed divergence between LeishIF4G-3 and its mammalian ortholog.Figure 3.

Bottom Line: LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E.A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27).The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.

ABSTRACT
Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.

Show MeSH
Related in: MedlinePlus