Abstract
The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein and is essential in all eukaryotes. However, the specific roles of eIF5A in translation and in other biological processes remain elusive. In the present study, we described the role of eIF5A, its posttranslational modifications (PTM), and the biosynthetic pathway needed for the PTM in Entamoeba histolytica, the protozoan parasite responsible for amoebic dysentery and liver abscess in humans. E. histolytica encodes two isotypes of eIF5A and two isotypes of enzymes, deoxyhypusine synthase (DHS), responsible for their PTM. Both of the two eIF5A isotypes are functional, whereas only one DHS (EhDHS1, but not EhDHS2), is catalytically active. The DHS activity increased ~2000-fold when EhDHS1 was co-expressed with EhDHS2 in Escherichia coli, suggesting that the formation of a heteromeric complex is needed for full enzymatic activity. Both EhDHS1 and 2 genes were required for in vitro growth of E. histolytica trophozoites, indicated by small antisense RNA-mediated gene silencing. In trophozoites, only eIF5A2, but not eIF5A1, gene was actively transcribed. Gene silencing of eIF5A2 caused compensatory induction of expression of eIF5A1 gene, suggesting interchangeable role of the two eIF5A isotypes and also reinforcing the importance of eIF5As for parasite proliferation and survival. Furthermore, using a sibling species, Entamoeba invadens, we found that eIF5A1 gene was upregulated during excystation, while eIF5A2 was downregulated, suggesting that eIF5A1 gene plays an important role during differentiation. Taken together, these results have underscored the essentiality of eIF5A and DHS, for proliferation and potentially in the differentiation of this parasite, and suggest that the hypusination associated pathway represents a novel rational target for drug development against amebiasis.
Highlights
Polyamines are low-molecular-weight nitrogenous bases that are essential for the regulation of cell growth and development [1]
Eukaryotic initiation factor 5A is a ubiquitous protein that is essential for cell proliferation
We found by small antisense RNA-mediated gene silencing that EhDHS1/2 and EheIF5A2 are essential for growth of E. histolytica trophozoites
Summary
Polyamines are low-molecular-weight nitrogenous bases that are essential for the regulation of cell growth and development [1]. Hypusine is an unusual amino acid [N (ε)- (4-amino2-hydroxybutyl)-lysine], which is uniquely synthesized on eIF5A at a specific lysine residue from spermidine by two catalytic steps [4]. This post-translational modification (PTM) in eukaryotes is achieved by the sequential reactions catalyzed by two enzymes: deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) (Fig 1) [4]. Hypusination is the most specific PTM known to date [5] and it is essential for eIF5A activity [6]. eIF5A is involved in elongation [7], termination [8], and stimulation of peptide bond formation [9], and it facilitates protein synthesis by resolving polyproline-induced ribosomal stalling; its role seems indispensable in synthesis of proline repeat-rich proteins [10]
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