Molecular Cloning and Characterization of a Protein Farnesyltransferase from the Enteric Protozoan Parasite Entamoeba histolytica

Abstract

Genes encoding ␣-and ␤-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the ␣-and ␤-subunit of E. histolytica FT (EhFT) were 298-and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant ␣-and ␤-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human HRas possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.

“Inject-Mix-React-Separate-and-Quantitate” (IMReSQ) Method for Screening Enzyme Inhibitors

“…this part of our study, we used the FTI-276 inhibitor that had been proven to inhibit EhFT. 4 When the concentration of E was zero, no P was formed and a single peak of S2 was detected ( Figure 2B, lower trace). In the presence of E but without I, S2 was converted in to P and, accordingly, two peaks were observed after P was separated from the remaining S2 ( Figure 2B, middle trace).…”

“…In this proof-of-principle work, we applied the method to study inhibition of recently cloned protein farnesyltransferase (FT) from parasite Entamoeba histolytica (Eh); this enzyme is a potential therapeutic target for antiparasitic drugs. 3,4 We identified three previously unknown inhibitors of EhFT and proved that IMReSQ could be used for accurately ranking the potencies of inhibitors.…”

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

“…This includes a subset of the active site residues, suggesting distinct specificity of binding to CaaX peptide substrates and inhibitors between PFT enzymes from these different species [15,16,18,40]. PGGT-I β orthologs have not been identified in gene databases of T. brucei and Leishmania species.…”

“…These are consistent with the CaaX specificity of the T. cruzi PGGT-I (Table1). Alterations in the residues involving substrate interactions in PFT and PGGT-I subunits between protozoan and mammalian orthologs may cause substantial differences in binding specificity for the CaaX motif and smallmolecule inhibitors in protozoa and mammalian cells [15][16][17][18][19][20][21]40]. This suggests not only different selectivity of inhibitors against parasite PFT and PGGT-I from that of mammalian enzymes but also differential consequences of inhibitory effects of PGGT-I or PFT on cellular events in the parasites from those in mammalian cells.…”

“…PFT has been found in pathogenic protozoan parasites including trypanosmatids (T. cruzi, T. brucei, and Leishmania), the malaria parasite (Plasmodium falciparum), and the enteric parasite Entamoeba histolytica [15][16][17][18]. PGGT-I has recently been found in E. histolytica [19].…”