title: 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 alpha subunit and a distinct beta 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 approximately 20% amino acid sequence identity to the PGGT-I beta 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 beta subunit of PGGT-I. Co-expression of this protein and the alpha 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 beta 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 approximately 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.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98195, USA. koheiy@u.washington.edu
authors.forename: Kohei
authors.initials: K
authors.lastname: Yokoyama
authors.forename: John R
authors.initials: JR
authors.lastname: Gillespie
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Van Voorhis
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Buckner
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003600:Cytosol / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011485:Protein Binding; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014316:Tritium / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D016054:DNA, Protozoan; D004791:Enzyme Inhibitors; D011106:Polyisoprenyl Phosphates; D015800:Protozoan Proteins; D014316:Tritium; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C002963:geranylgeranyl pyrophosphate