LeishIF4E-5 Is a Promastigote-Specific Cap-Binding Protein in Leishmania

Abstract

Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular promastigotes that reside in the vectors’ alimentary canal to obligatory intracellular non-motile amastigotes that are harbored by macrophages of the mammalian hosts. The transition between vector and host exposes them to a broad range of environmental conditions that induces a developmental program of gene expression, with translation regulation playing a key role. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E. All six isoforms show a relatively low degree of conservation with eIF4Es of other eukaryotes, as well as among themselves. This variability could suggest that they have been assigned discrete roles that could contribute to their survival under the changing environmental conditions. Here, we describe LeishIF4E-5, a LeishIF4E paralog. Despite the low sequence conservation observed between LeishIF4E-5 and other LeishIF4Es, the three aromatic residues in its cap-binding pocket are conserved, in accordance with its cap-binding activity. However, the cap-binding activity of LeishIF4E-5 is restricted to the promastigote life form and not observed in amastigotes. The overexpression of LeishIF4E-5 shows a decline in cell proliferation and an overall reduction in global translation. Immuno-cytochemical analysis shows that LeishIF4E-5 is localized in the cytoplasm, with a non-uniform distribution. Mass spectrometry analysis of proteins that co-purify with LeishIF4E-5 highlighted proteins involved in RNA metabolism, along with two LeishIF4G paralogs, LeishIF4G-1 and LeishIF4G-2. These vary in their conserved eIF4E binding motif, possibly suggesting that they can form different complexes.

Characterization of an Atypical eIF4E Ortholog in Leishmania, LeishIF4E-6

“…Further confirmation that these multiple bands were derived from LeishIF4G-5 were obtained from mass spectrometry analysis performed on each of the cleavage products ( Figure S4A,B and Table S3 ). Cleavage of proteins that are part of the translation apparatus is common in Leishmania , as previously discussed [ 24 , 39 ].…”

“…These Trp residues are conserved in most eIF4Es but occasionally are replaced with other aromatic residues, such as Tyr or Phe. However, the substitution of conserved Trp residues may influence their binding of m 7 GTP cap, as observed for LeishIF4E-5, where the Trp residue that is analogous to the mouse Trp102 is replaced with Tyr [ 24 ]. Other residues may also assist the cap-binding activity, as basic residues within the pocket can interact with the acidic backbone of the cap structure.…”

“…The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E and five paralogs of eIF4G [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. While the yeast genome encodes a single eIF4E that is essential for viability and two eIF4G homologs [ 30 , 31 ], mammalian cells encode for three eIF4Es and two eIF4Gs isoforms.…”

“…Overexpression of LeishIF4E-5 showed a decline in cell proliferation and an overall reduction in global translation. Thus, LeishIF4E-5 could be a putative translational repressor [ 24 ].…”

Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5 and EIF4E6 inTrypanosoma brucei: EIF4E2 maintains the abundances of S-phase mRNAs

“…The T. brucei proteins are now known to be just as abundant as EIF4E3 and EIF4E4 (Dejung et al ., 2016, Tinti & Ferguson, 2022)(Supplementary Table S1). EIF4E5 is associated with EIF4G1 and EIF4G2 in both T. brucei and Leishmania (Freire et al ., 2014b, Shrivastava et al ., 2021). In T. brucei , it was also associated with a dynein light chain (LC8), a putative capping enzyme, the putative RNA-binding protein RBP43, the two 14-3-3 protein paralogues, EF1, and two other proteins of unknown function (Tb927.8.4560, Tb927.11.6010) (Freire et al ., 2014b).…”