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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 and LeishIF4A-1 copurifiy with TAP-tagged LeishIF4E-1 and LeishIF4E-4. Leishmania major wild-type or transgenic cells expressing TAP-tagged LeishIF4E-1 (A) and LeishIF4E-4 (B) were lyzed and the soluble supernatant (S) was loaded on streptavidin-Sepharose beads. The beads were washed, eluted with biotin and repurified in tandem over IgG-Sepharose beads (LeishIF4E-1) or over m7GTP-Sepharose beads (LeishIF4E-4). The corresponding beads were washed (W) and eluted (E, see Materials and Methods section). The W and E lanes were loaded with 20% of the wash and eluate volumes, respectively. All proteins were fractionated by SDS–PAGE (12%) and subjected to western blot analysis using antibodies specific against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1.
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Figure 5: LeishIF4G-3 and LeishIF4A-1 copurifiy with TAP-tagged LeishIF4E-1 and LeishIF4E-4. Leishmania major wild-type or transgenic cells expressing TAP-tagged LeishIF4E-1 (A) and LeishIF4E-4 (B) were lyzed and the soluble supernatant (S) was loaded on streptavidin-Sepharose beads. The beads were washed, eluted with biotin and repurified in tandem over IgG-Sepharose beads (LeishIF4E-1) or over m7GTP-Sepharose beads (LeishIF4E-4). The corresponding beads were washed (W) and eluted (E, see Materials and Methods section). The W and E lanes were loaded with 20% of the wash and eluate volumes, respectively. All proteins were fractionated by SDS–PAGE (12%) and subjected to western blot analysis using antibodies specific against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1.

Mentions: In view of the different results obtained in the in vitro and in vivo tagging experiments that examine the interaction between LeishIF4G-3 and LeishIF4E-1, a reciprocal in vivo tagging approach was used. LeishIF4E-1 and LeishIF4E-4 were fused to the tandem affinity purification (TAP)-tag, using the pPNSAP1 vector (33), which adds two different in frame tags, a Protein A peptide and a streptavidin-binding peptide. The tagged LeishIF4E-1 was first bound to streptavidin beads, and the biotin-eluted fractions were further subjected to purification on an IgG-Sepharose column (Figure 5A). For the tagged LeishIF4E-4, the second round of purification was performed over m7GTP-Sepharose, taking advantage of the cap-binding activity of LeishIF4E-4 (Figure 5B). Western blot analysis of the purified complexes confirmed that LeishIF4G-3 was coprecipitated with LeishIF4E-1 and LeishIF4E-4. The pull-down of LeishIF4G-3 by the tagged LeishIF4E-1 was more efficient than the reciprocal pull-down of LeishIF4E-1 by tagged LeishIF4G-3, possibly due to over expression of the tagged LeishIF4E-1 in the transgenic parasites, which could serve to enhance a basically weaker interaction. The two TAP-tagged cap-binding complexes also contained LeishIF4A-1, the third components of the eIF4F complex.Figure 5.


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 and LeishIF4A-1 copurifiy with TAP-tagged LeishIF4E-1 and LeishIF4E-4. Leishmania major wild-type or transgenic cells expressing TAP-tagged LeishIF4E-1 (A) and LeishIF4E-4 (B) were lyzed and the soluble supernatant (S) was loaded on streptavidin-Sepharose beads. The beads were washed, eluted with biotin and repurified in tandem over IgG-Sepharose beads (LeishIF4E-1) or over m7GTP-Sepharose beads (LeishIF4E-4). The corresponding beads were washed (W) and eluted (E, see Materials and Methods section). The W and E lanes were loaded with 20% of the wash and eluate volumes, respectively. All proteins were fractionated by SDS–PAGE (12%) and subjected to western blot analysis using antibodies specific against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1.
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Figure 5: LeishIF4G-3 and LeishIF4A-1 copurifiy with TAP-tagged LeishIF4E-1 and LeishIF4E-4. Leishmania major wild-type or transgenic cells expressing TAP-tagged LeishIF4E-1 (A) and LeishIF4E-4 (B) were lyzed and the soluble supernatant (S) was loaded on streptavidin-Sepharose beads. The beads were washed, eluted with biotin and repurified in tandem over IgG-Sepharose beads (LeishIF4E-1) or over m7GTP-Sepharose beads (LeishIF4E-4). The corresponding beads were washed (W) and eluted (E, see Materials and Methods section). The W and E lanes were loaded with 20% of the wash and eluate volumes, respectively. All proteins were fractionated by SDS–PAGE (12%) and subjected to western blot analysis using antibodies specific against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1.
Mentions: In view of the different results obtained in the in vitro and in vivo tagging experiments that examine the interaction between LeishIF4G-3 and LeishIF4E-1, a reciprocal in vivo tagging approach was used. LeishIF4E-1 and LeishIF4E-4 were fused to the tandem affinity purification (TAP)-tag, using the pPNSAP1 vector (33), which adds two different in frame tags, a Protein A peptide and a streptavidin-binding peptide. The tagged LeishIF4E-1 was first bound to streptavidin beads, and the biotin-eluted fractions were further subjected to purification on an IgG-Sepharose column (Figure 5A). For the tagged LeishIF4E-4, the second round of purification was performed over m7GTP-Sepharose, taking advantage of the cap-binding activity of LeishIF4E-4 (Figure 5B). Western blot analysis of the purified complexes confirmed that LeishIF4G-3 was coprecipitated with LeishIF4E-1 and LeishIF4E-4. The pull-down of LeishIF4G-3 by the tagged LeishIF4E-1 was more efficient than the reciprocal pull-down of LeishIF4E-1 by tagged LeishIF4G-3, possibly due to over expression of the tagged LeishIF4E-1 in the transgenic parasites, which could serve to enhance a basically weaker interaction. The two TAP-tagged cap-binding complexes also contained LeishIF4A-1, the third components of the eIF4F complex.Figure 5.

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