Characterization of the deoxyhypusine synthases and eukaryotic translation initiation factor 5A of Entamoeba

Entamoeba histolytica infects approximately 50 million people worldwide and causes 70-100,000 deaths annually. Disease transmission is dependent on ingestion of the infectious cyst stage of the parasite, making it critical to understand the cellular mechanisms of stage conversion within the host environment. Recent gene expression studies showed that treatment of E. histolytica with the potent deacetylase inhibitor Trichostatin A (TSA) caused hyperacetylation of histones and up-regulation of a subset of genes that include those associated with cyst formation. One of the upregulated genes encodes deoxyhypusine synthase, a protein involved in the modification of the eukaryotic translation initiation factor 5A (eIF5A). The roles of eIF5A and DHS have not been established in Entamoeba but eIF5A has been implicated in translation initiation/elongation, mRNA regulation, and regulation of cell proliferation in higher eukaryotes. The putative roles of these proteins in higher eukaryotes, in combination with the gene expression data from Entamoeba, led to the hypothesis that these proteins could play a role in the post-translational regulation of amoebic differentiation. The Entamoeba genome was found to encode two families of eIF5A proteins. We also identified two deoxyhypusine synthase sequences in the E. histolytica genome, EhDHS77 and EhDHS83. Both EhDHS77 and EhDHS83 exhibited a high degree of sequence conservation of critical residues, but EhDHS83 lacks the active site lysine residue. Despite retention of the active site lysine, when expressed individually EhDHS77 has very poor catalytic activity, whereas EhDHS83 is completely inactive. When co-expressed (as EhDual DHS), EhDHS77 and EhDHS83 yielded a protein complex with high levels of catalytic activity. Size exclusion chromatography indicated that the enzyme is a heterotetramer consisting of ∼42 KDa subunits. The DHS inhibitor N1-guanyl-1,7-diaminoheptane (GC7) inhibited EhDual DHS activity in a competitive manner in vitro, and affected growth of cultured E. histolytica trophozoites