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.
Highlights
Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular flagellated and motile promastigotes in the female sand flies to obligatory intracellular non-motile amastigotes that reside within macrophages of the mammalian hosts
The L. amazonensis LeishIF4E-5 and its orthologs from T. brucei and from different non-pathogenic kinetoplastids (Leptomonas seymouri and Crithidia fasciulata) have aromatic residues at parallel positions: in L. amazonensis, the Trp residues are found at positions 32 and 151, and a Tyr is at position 83
The peptides generated We examined thfreomLtehieshpuIFll4-dEo-w5nionfttehreaScBtPin-tgaggperdoLteeiisnhsIF4bEy-5pwuerlel-iddoenwtifinedexbapseerdimonethnetsanfnoolt-ated lowed by mass spectLtrho. emmSaBejoPtrr-pytargoagtneeiadnlsyluisnciisTfe.rriTTarshyee.pDTphBre.eCisdeoennntctrioefileopdfuplLlr-eodtioeswihnnIsFsw4weEer-ree5spuienbrjfetochrtemedeedtlouostnteacdteislftlsircaeacxl ptairnoeanslsyisnigs was verified by WestuesrinngaPnearsleyussissouftswinarge aanndtipbrootdeiinesswaigthaainLsotg2thfoeldSeBnPri-cthamge.nTt hofe1.p6e(tphtriede-efosldgechna-nge) erated from the pull-down of the Streptavidin binding peptide (SBP)-tagged LeishIF4E-5 were identified based on the annotated L. major proteins in TriTrypDB
Summary
Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular flagellated and motile promastigotes in the female sand flies to obligatory intracellular non-motile amastigotes that reside within macrophages of the mammalian hosts. The alternation between the sandfly vector and mammalian host is as an integral part of their life cycle These transitions between two different hosts are accompanied by shifts in environmental conditions, mainly, extreme changes in temperature and pH [1]. LeishIF4E-4 is considered to be a canonical initiation factor as it binds efficiently to the cap-4 structure, which is the unique mRNA cap in trypanosomatids [8,9], and associates with LeishIF4G-3, one of the five LeishIF4G candidates [10]. Under conditions that induce differentiation to axenic amastigotes, LeishIF4E-4 loses its cap-binding activity [11] Another cap-binding factor, LeishIF4E-1, binds cap-4 efficiently [12], but it does not associate with any of the LeishIF4G candidates. These experiments highlight that LeishIF4E-5 associates with proteins related to RNA metabolism as well as with other LeishIF4Es
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