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et al. could inhibit the cap-binding activity of LeishIF4Es. Using a functional assay, we show that a recombinant form of Leish4E-IP2 inhibits the cap-binding activity of LeishIF4E-1 and LeishIF4E-3. Furthermore, we show that transgenic parasites expressing a tagged version of Leish4E-IP2 also display reduced cap-binding activities of tested LeishIF4Es, and decreased global Rabbit polyclonal to ZNF490 translation. Given its ability to bind more than a single LeishIF4E, we suggest that Leish4E-IP2 could serve as a broad-range repressor of protein synthesis. INTRODUCTION parasites cycle between invertebrate vectors and mammalian hosts. In doing so, they differentiate from flagellated promastigotes residing in the intestinal tract of sand-flies, into non-flagellated amastigotes, which are obligatory intracellular forms of the parasites. Amastigotes exist within phagolysosomal vacuoles of macrophages and other cells of the immune system. During their life cycle, a developmental program of gene expression enables the parasites to adapt to different environmental conditions, including temperature, pH and variations in nutrient supplies. Translation regulation plays a key role in driving this program, especially in CI 972 the absence of conventional transcription activation mechanisms (1C3). In Opisthokonts, cap-dependent translation initiation is the default pathway for protein synthesis. The translational initiation complex assembles on the 5 cap (m7GTP) of messenger RNAs (mRNAs) through the eukaryotic initiation factor 4F complex (eIF4F). eIF4F comprises the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A, and the scaffold protein eIF4G. eIF4G binds eIF3, which recruits the small ribosomal subunit. eIF4G also interacts with eIF4E, through a consensus binding motif, Y(X)4L (where X is any amino acid and is a hydrophobic residue). Protein synthesis can be inhibited by the binding of hypo-phosphorylated 4E-BPs to eIF4E. 4E-BP also contains a Y(X)4L motif (4) and competes with eIF4G on interacting with eIF4E, thus blocking the formation of the eIF4E/eIF4G complex (5,6). Since the identification of 4E-BP1, many other eIF4E regulatory proteins have been identified in several organisms. Translation regulation is a central mechanism that drives the developmental program of gene expression in trypanosomatids. This is especially emphasized given their unusual way of generating matured mRNAs (1,7,8). Transcription of primary mRNAs is polycistronic, and there is no evidence for any conventional transcription activation mechanisms of mRNAs. The polycistronic transcripts are further processed to mature monocistronic mRNAs via trans-splicing and polyadenylation (9,10). Since digenetic parasites, such as and Trypanosomes encode six paralogs of eIF4E (LeishIF4Es) and at least five paralogs of eIF4G (LeishIF4Gs). These contain a conserved MIF4G domain (11C14) and the consensus Y(X)4L element, except LeishIF4G-4 which lacks this motif, despite its strong interaction with LeishIF4E-3 (15). LeishIF4E-1 through ?4 were intensively studied both in and Trypanosomes (16C21). Two additional orthologs of eIF4E, TbIF4E-5 and TbIF4E-6 were identified in (22,23), and their orthologs were subsequently found in the genomes. The high number of eIF4E and eIF4G orthologs in and Trypanosomes could coincide with the need of these organisms to survive under extreme conditions at a specific given point during CI 972 their life cycle. Understanding the roles of these multiple isoforms remains a CI 972 challenging goal (24). LeishIF4E-4 is generally accepted to be a canonical translation initiation factor in promastigotes, based on its efficient cap-binding activity and its ability to anchor a functional cap-binding complex, including LeishIF4G-3 and LeishIF4A-1 (25,26). LeishIF4E-4 has a non-conserved N-terminal extension, which contains multiple phosphorylation sites (27). This LeishIF4E-4 N-terminus also binds LeishPABP1, unlike in other eukaryotes, where eIF4G is responsible for this interaction (20,28,29). Exposure CI 972 of to mammalian-level temperatures (amastigotes stage) eliminates its cap-binding activity, as well as its binding to LeishIF4G-3. Under these conditions, the isoform LeishIF4E-1 binds efficiently to the cap, suggesting that this protein plays a role in both life stages (20). Unlike these two eIF4E orthologs, LeishIF4E-3 binds inefficiently to the.