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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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Leishmania major cap-binding complex purification. Leishmania major cells were lyzed and the soluble protein extract (S) was loaded over an m7GTP-Sepharose column. After collection of the flowthrough (FT) the column was washed (W) with CB (see Materials and Methods section) and with CB containing 100 μM GTP (GTP). The proteins were eluted (E) with CB containing 500 mM NaCl. Equal samples from the different fractions were separated by SDS–PAGE and analyzed by western blot using specific antibodies against the different subunits of the Leishmania cap-binding complex (LeishIF4E-1 through LeishIF4E-4, LeishIF4G-3, LeishIF4A-1). Similar results were obtained when the m7GTP-Sepharose column was eluted with free m7GTP.
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Figure 1: Leishmania major cap-binding complex purification. Leishmania major cells were lyzed and the soluble protein extract (S) was loaded over an m7GTP-Sepharose column. After collection of the flowthrough (FT) the column was washed (W) with CB (see Materials and Methods section) and with CB containing 100 μM GTP (GTP). The proteins were eluted (E) with CB containing 500 mM NaCl. Equal samples from the different fractions were separated by SDS–PAGE and analyzed by western blot using specific antibodies against the different subunits of the Leishmania cap-binding complex (LeishIF4E-1 through LeishIF4E-4, LeishIF4G-3, LeishIF4A-1). Similar results were obtained when the m7GTP-Sepharose column was eluted with free m7GTP.

Mentions: A bioinformatics search of the Leishmania and trypanosome genomes highlighted several proteins that share structural domains with eIF4G from higher eukaryotes. To confirm which of the candidates is indeed part of the translation initiation machinery, functional assays were pursued. In higher eukaryotes, components of the eIF4F complex can be eluted from an m7GTP-Sepharose column through the ability of eIF4E to interact with the m7GTP cap structure. Based on former results showing that specific recombinant LeishIF4E isoforms can interact with m7GTP, components of the endogenous cap-binding complex were also identified by affinity purification of proteins from whole cell extracts of L. major over an m7GTP-Sepharose column. The parasite cap-binding complexes were eluted and proteins were separated by SDS–PAGE. A ∼70 kDa excised band (Supplementary Figure 1A) was identified in two independent experiments by mass spectrometry as LmjF16.1600, a homolog of eIF4G that was denoted LeishIF4G-3 (Supplementary Figure 1B). This finding supports that LeishIF4G-3 is a potential candidate to act as a translational initiation factor and is in good agreement with a former report showing that this protein can interact with the parasite eIF4A helicase, which is one of the eIF4F subunits (32). Western blot analysis of the fractions that were eluted from the m7GTP-Sepharose column depicted the presence of LeishIF4E-1 and LeishIF4E-4, LeishIF4A as well as LeishIF4G-3 (Figure 1). LeishIF4E-2 and LeishIF4E-3 were not expected to be eluted from the m7GTP column. LeishIF4E-2 does not bind efficiently to m7GTP, and LeishIF4E-3 does not purify well from m7GTP-Sepharose, although it was shown to bind m7GTP using fluorescence titration assays (31,32). Thus, a probable form of the LeishIF4F complex which includes LeishIF4E-1 or LeishIF4E-4, LeishIF4A-1 and LeishIF4G-3 was purified over the m7GTP-Sepharose column.Figure 1.


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)

Leishmania major cap-binding complex purification. Leishmania major cells were lyzed and the soluble protein extract (S) was loaded over an m7GTP-Sepharose column. After collection of the flowthrough (FT) the column was washed (W) with CB (see Materials and Methods section) and with CB containing 100 μM GTP (GTP). The proteins were eluted (E) with CB containing 500 mM NaCl. Equal samples from the different fractions were separated by SDS–PAGE and analyzed by western blot using specific antibodies against the different subunits of the Leishmania cap-binding complex (LeishIF4E-1 through LeishIF4E-4, LeishIF4G-3, LeishIF4A-1). Similar results were obtained when the m7GTP-Sepharose column was eluted with free m7GTP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Leishmania major cap-binding complex purification. Leishmania major cells were lyzed and the soluble protein extract (S) was loaded over an m7GTP-Sepharose column. After collection of the flowthrough (FT) the column was washed (W) with CB (see Materials and Methods section) and with CB containing 100 μM GTP (GTP). The proteins were eluted (E) with CB containing 500 mM NaCl. Equal samples from the different fractions were separated by SDS–PAGE and analyzed by western blot using specific antibodies against the different subunits of the Leishmania cap-binding complex (LeishIF4E-1 through LeishIF4E-4, LeishIF4G-3, LeishIF4A-1). Similar results were obtained when the m7GTP-Sepharose column was eluted with free m7GTP.
Mentions: A bioinformatics search of the Leishmania and trypanosome genomes highlighted several proteins that share structural domains with eIF4G from higher eukaryotes. To confirm which of the candidates is indeed part of the translation initiation machinery, functional assays were pursued. In higher eukaryotes, components of the eIF4F complex can be eluted from an m7GTP-Sepharose column through the ability of eIF4E to interact with the m7GTP cap structure. Based on former results showing that specific recombinant LeishIF4E isoforms can interact with m7GTP, components of the endogenous cap-binding complex were also identified by affinity purification of proteins from whole cell extracts of L. major over an m7GTP-Sepharose column. The parasite cap-binding complexes were eluted and proteins were separated by SDS–PAGE. A ∼70 kDa excised band (Supplementary Figure 1A) was identified in two independent experiments by mass spectrometry as LmjF16.1600, a homolog of eIF4G that was denoted LeishIF4G-3 (Supplementary Figure 1B). This finding supports that LeishIF4G-3 is a potential candidate to act as a translational initiation factor and is in good agreement with a former report showing that this protein can interact with the parasite eIF4A helicase, which is one of the eIF4F subunits (32). Western blot analysis of the fractions that were eluted from the m7GTP-Sepharose column depicted the presence of LeishIF4E-1 and LeishIF4E-4, LeishIF4A as well as LeishIF4G-3 (Figure 1). LeishIF4E-2 and LeishIF4E-3 were not expected to be eluted from the m7GTP column. LeishIF4E-2 does not bind efficiently to m7GTP, and LeishIF4E-3 does not purify well from m7GTP-Sepharose, although it was shown to bind m7GTP using fluorescence titration assays (31,32). Thus, a probable form of the LeishIF4F complex which includes LeishIF4E-1 or LeishIF4E-4, LeishIF4A-1 and LeishIF4G-3 was purified over the m7GTP-Sepharose column.Figure 1.

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