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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 comigrates with LeishIF4E-1 and LeishIF4E-4 by sucrose gradients analysis. Extracts of L. major cells grown at 26°C, and after exposure to 35°C for 18 h were loaded on 10–50% sucrose gradients. (A) Pattern of polysome migration on sucrose gradients. Fractions were collected from the top of the gradient and the O.D260nm was monitored. (26°C, continuous line; 35°C, broken line). Proteins were recovered by TCA precipitation, and immunoblotted with antibodies raised against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 as well as with anti-rpS6, for detection of fractions that contain complexes containing 40S particles. Western analysis was done on fractions of cells that were grown at 26°C (B) and 35°C (C).
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Figure 2: LeishIF4G-3 comigrates with LeishIF4E-1 and LeishIF4E-4 by sucrose gradients analysis. Extracts of L. major cells grown at 26°C, and after exposure to 35°C for 18 h were loaded on 10–50% sucrose gradients. (A) Pattern of polysome migration on sucrose gradients. Fractions were collected from the top of the gradient and the O.D260nm was monitored. (26°C, continuous line; 35°C, broken line). Proteins were recovered by TCA precipitation, and immunoblotted with antibodies raised against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 as well as with anti-rpS6, for detection of fractions that contain complexes containing 40S particles. Western analysis was done on fractions of cells that were grown at 26°C (B) and 35°C (C).

Mentions: The LeishIF4G-3 ORF is smaller than the mammalian eIF4G-I and contains 635 compared with 1560 amino acids, respectively. It includes the middle domain of eIF4G (MIF4G), which is highly conserved among divergent organisms (see Supplementary Figure 2), but the other regions of LeishIF4G-3 bear no significant sequence similarity to the human eIF4GI, with a rather short N-terminus. In higher eukaryotes, eIF4G was shown to be mostly distributed in the cytoplasm (37), this was verified in Leishmania as well (data not shown). To investigate whether LeishIF4G-3 comigrates with LeishIF4E-1 and LeishIF4E-4 in particles of the same size, cell extracts of L. major cells that were grown at 26°C, a temperature used for growth of Leishmania promastigotes, were fractionated over sucrose gradients. The polysome profile was monitored by RNA absorbance at 260 nm (Figure 2A) and migration of the ribosomal small subunit was monitored by western analysis using antibodies directed against rpS6. Migration of the different LeishIF4F subunits were also monitored by western analysis. As shown in Figure 2B, LeishIF4G-3 comigrates with LeishIF4E-1 and LeishIF4E-4 in fractions that contain free RNPs at the top of the gradient (up to fraction 10), as well as in heavier fractions (10–14) that contain the 40S subunits, as demonstrated by their reaction with anti-rpS6 antibodies. In addition, for each of the proteins examined, a weaker reaction was observed in fractions that correspond to the 80S particles (fractions 23–25). However, this fraction highlighted lower bands of LeishIF4E-4 and LeishIF4G-3 as well as a higher band of LeishIF4E-1. Given that the antibodies against LeishIF4E-1 and LeishIF4E-4 do not cross-react with the other eIF4E paralogs in Leishmania, these bands may represent different modification forms of the protein, possibly caused by phosphorylation. A very weak association of LeishIF4E-1 and LeishIF4E-4 with polysomes was observed (fractions 27–35).Figure 2.


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 comigrates with LeishIF4E-1 and LeishIF4E-4 by sucrose gradients analysis. Extracts of L. major cells grown at 26°C, and after exposure to 35°C for 18 h were loaded on 10–50% sucrose gradients. (A) Pattern of polysome migration on sucrose gradients. Fractions were collected from the top of the gradient and the O.D260nm was monitored. (26°C, continuous line; 35°C, broken line). Proteins were recovered by TCA precipitation, and immunoblotted with antibodies raised against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 as well as with anti-rpS6, for detection of fractions that contain complexes containing 40S particles. Western analysis was done on fractions of cells that were grown at 26°C (B) and 35°C (C).
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Figure 2: LeishIF4G-3 comigrates with LeishIF4E-1 and LeishIF4E-4 by sucrose gradients analysis. Extracts of L. major cells grown at 26°C, and after exposure to 35°C for 18 h were loaded on 10–50% sucrose gradients. (A) Pattern of polysome migration on sucrose gradients. Fractions were collected from the top of the gradient and the O.D260nm was monitored. (26°C, continuous line; 35°C, broken line). Proteins were recovered by TCA precipitation, and immunoblotted with antibodies raised against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 as well as with anti-rpS6, for detection of fractions that contain complexes containing 40S particles. Western analysis was done on fractions of cells that were grown at 26°C (B) and 35°C (C).
Mentions: The LeishIF4G-3 ORF is smaller than the mammalian eIF4G-I and contains 635 compared with 1560 amino acids, respectively. It includes the middle domain of eIF4G (MIF4G), which is highly conserved among divergent organisms (see Supplementary Figure 2), but the other regions of LeishIF4G-3 bear no significant sequence similarity to the human eIF4GI, with a rather short N-terminus. In higher eukaryotes, eIF4G was shown to be mostly distributed in the cytoplasm (37), this was verified in Leishmania as well (data not shown). To investigate whether LeishIF4G-3 comigrates with LeishIF4E-1 and LeishIF4E-4 in particles of the same size, cell extracts of L. major cells that were grown at 26°C, a temperature used for growth of Leishmania promastigotes, were fractionated over sucrose gradients. The polysome profile was monitored by RNA absorbance at 260 nm (Figure 2A) and migration of the ribosomal small subunit was monitored by western analysis using antibodies directed against rpS6. Migration of the different LeishIF4F subunits were also monitored by western analysis. As shown in Figure 2B, LeishIF4G-3 comigrates with LeishIF4E-1 and LeishIF4E-4 in fractions that contain free RNPs at the top of the gradient (up to fraction 10), as well as in heavier fractions (10–14) that contain the 40S subunits, as demonstrated by their reaction with anti-rpS6 antibodies. In addition, for each of the proteins examined, a weaker reaction was observed in fractions that correspond to the 80S particles (fractions 23–25). However, this fraction highlighted lower bands of LeishIF4E-4 and LeishIF4G-3 as well as a higher band of LeishIF4E-1. Given that the antibodies against LeishIF4E-1 and LeishIF4E-4 do not cross-react with the other eIF4E paralogs in Leishmania, these bands may represent different modification forms of the protein, possibly caused by phosphorylation. A very weak association of LeishIF4E-1 and LeishIF4E-4 with polysomes was observed (fractions 27–35).Figure 2.

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