Protein geranylgeranyltransferase-I of Trypanosoma cruzi

Abstract

Protein geranylgeranyltransferase type I (PGGT-I) and protein farnesyltransferase (PFT) occur in many eukaryotic cells. Both consist of two subunits, the common αsubunit and a distinct β subunit. In the gene database of protozoa Trypanosoma cruzi, the causative agent of Chagas' disease, a putative protein that consists of 401 amino acids with ∼20% amino acid sequence identity to the PGGT-I β of other species was identified, cloned, and characterized. Multiple sequence alignments show that the T. cruzi ortholog contains all three of the zinc-binding residues and several residues uniquely conserved in the β subunit of PGGT-I. Co-expression of this protein and the α subunit of T. cruzi PFT in Sf9 insect cells yielded a dimeric protein that forms a tight complex selectively with [ 3 H] geranylgeranyl pyrophosphate, indicating a key characteristic of a functional PGGT-I. Recombinant T. cruzi PGGT-I ortholog showed geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Most of the CaaX-containing proteins with X=Leu are good substrates of T. cruzi PGGT-I, and those with X=Met are substrates for both T. cruzi PFT and PGGT-I, whereas unlike mammalian PGGT-I, those with X=Phe are poor substrates for T. cruzi PGGT-I. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif are found in the T. cruzi gene database. Among five C-terminal peptides of those tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi PGGT-I. Other peptides with CTQQ (Tcj2 DNAJ protein), CAVM (TcPRL-1 protein tyrosine phosphatase), CHFM (a small GTPase like protein), and CQLF (TcRho1 GTPase) were specific substrates for T. cruzi PFT but not for PGGT-I. The mRNA and protein of the T. cruzi PGGT-I β ortholog were detected in three life-cycle stages of T. cruzi. Cytosol fractions from trypomastigotes (infectious mammalian stage) and epimastigotes (insect stage) were shown to contain levels of PGGT-I activity that are ∼100-fold lower than PFT activity. The CaaX mimetics known as PGGT-I inhibitors show very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, suggesting the potential to develop selective inhibitors against the parasite enzyme.

Targeting protein prenylation for cancer therapy

“…FTIs specifically designed to inhibit parasitic FT and not mammalian FT are significantly more toxic to parasitic protozoa 194,195 . Recently, a GGT1 from Trypanosoma cruzi , the parasite responsible for Chagas disease, was cloned 196 , and GGTIs may also be effective against these diseases.…”

Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi

“…Therefore, TcRab32 is specifically geranylgeranylated. Previous studies of recombinant T. cruzi protein geranylgeranyl transferase I (GGTI) using a panel of mammalian and yeast protein substrates report that two mammalian Rab-family GTPases containing the C-terminal CXC sequence do not serve as substrates for this enzyme, as expected (Yokoyama et al, 2008;Nepomuceno-Silva et al, 2001). Accordingly, Prenylation Prediction Suite (PrePS) predicts that geranylgeranyl transferase II (GGTII) is the enzyme involved in the prenylation of this protein.…”

Thiol-Based Posttranslational Modifications in Parasites

“…show a different substrate specificity toward the CaaX motif compared to mammalian farnesyltransferases due to distinct amino acids in the substrate binding pocket, and have several insertions with unknown function (32, 33, 250). Moreover, T. cruzi employs a type I geranylgeranyltransferase with a distinct CaaX specificity that is exploited for the development of selective inhibitors (249,250). Imidazole-containing CaaX peptidomimetics inhibit the growth of T. brucei, with the effect being attributed to inhibition of T. brucei farnesyltransferase (160,161).…”