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: Kohei Yokoyama, John R Gillespie, Wesley C Van Voorhis, Frederick S Buckner, Michael H Gelb
• Journal: Molecular and biochemical parasitology (Mol Biochem Parasitol) Vol. 157 Issue 1 Pg. 32-43 (Jan 2008) ISSN: 0166-6851 [Print] Netherlands
• PMID: 17996962 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• DNA, Protozoan
• Enzyme Inhibitors
• Polyisoprenyl Phosphates
• Protozoan Proteins
• Tritium
• Alkyl and Aryl Transferases
• geranylgeranyltransferase type-I
• geranylgeranyl pyrophosphate

Topics

• Alkyl and Aryl Transferases (antagonists & inhibitors, genetics, isolation & purification, metabolism)
• Amino Acid Sequence
• Animals
• Binding Sites
• Cloning, Molecular
• Conserved Sequence
• Cytosol (chemistry)
• DNA, Protozoan (chemistry, genetics)
• Enzyme Inhibitors (pharmacology)
• Isotope Labeling
• Molecular Sequence Data
• Polyisoprenyl Phosphates (metabolism)
• Protein Binding
• Protozoan Proteins (antagonists & inhibitors, genetics, isolation & purification, metabolism)
• Sequence Alignment
• Substrate Specificity
• Tritium (metabolism)
• Trypanosoma cruzi (enzymology)