Cloning, Heterologous Expression, and Distinct Substrate Specificity of Protein Farnesyltransferase from Trypanosoma brucei

Buckner, Frederick S.; Yokoyama, Kohei; Nguyen, Lan Thi; Grewal, Anita; Erdjument‐Bromage, Hediye; Tempst, Paul; Strickland, Corey L.; Xiao, Li; Voorhis, Wesley C. Van; Gelb, Michael H.

doi:10.1074/jbc.m000975200

Cited by 55 publications

(44 citation statements)

References 29 publications

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“…FTIs are cytotoxic to these protozoa, perhaps because they contain PFT but seem to lack PGGT-I (refs. [133][134][135][136] ). In the case of malaria, the target of FTI toxicity is almost certainly PFT, given that parasites that have become resistant to FTIs contain mutant PFTs that bind FTIs less tightly 137 .…”

Section: Ftis As Tropical Parasitic Disease Therapeuticsmentioning

confidence: 99%

Therapeutic intervention based on protein prenylation and associated modifications

et al. 2006

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In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.Isoprenoids are lipids made of five-carbon blocks, and they constitute one of the main classes of natural products. A subset of isoprenoids called prenyl groups are found on a variety of biological substances, including proteins. Prenylated proteins and peptides are found in most (if not all) eukaryotes. They arise from the post-translational attachment of 15-carbon farnesyl or 20-carbon geranylgeranyl groups to the C-terminal segment of proteins. Protein prenyl groups not only are hydrophobic elements that bind proteins to membranes, but, at least in some cases, they also function as molecular handles that bind to hydrophobic grooves on the surface of soluble protein factors; these factors then remove the prenylated protein from the membrane in a regulated manner. NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2010 June 28. Published in final edited form as:Nat Chem Biol. 2006 October ; 2(10): 518-528. doi:10.1038/nchembio818. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptProtein prenylation has been vigorously studied over the past ~15 years because it is found on several signaling proteins (including heterotrimeric G proteins) that connect cell surface receptors to intracellular effectors, and also on Ras proteins, one of the most common oncoproteins found in human tumors. Ras proteins serve as molecular switches at cellular membranes to control propagation of growth signals from cell surface receptors to nuclear transcription factors. Because Ras mutations are important in many human cancers, there has been extensive focus on Ras prenylation. Discovery of protein prenyl groups and structural varietiesThe first reports of prenylated proteins and peptides described the secreted pheromone peptides from jelly fungi 1,2 , whose structure resembles that of the well-known a-factor mating pheromone from baker's yeast (Saccharomyces cerevisiae), which contains a cysteine methylester farnesylated at the C terminus 3 . In the 1980s, studies on the timing of cholesterol biosynthesis with respect to the cell cycle in human cells led to the discovery that a compound deriv...

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Section: Ftis As Tropical Parasitic Disease Therapeuticsmentioning

confidence: 99%

Therapeutic intervention based on protein prenylation and associated modifications

et al. 2006

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“…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.…”

Section: Inhibitor Studiesmentioning

confidence: 99%

“…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.…”

Section: Inhibitor Studiesmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

“…Effective PFT inhibitors to block growth of T. cruzi amastigotes in mammalian host cells have not been found, although the growth of T. cruzi showed significantly more sensitivity to PFT inhibition than the mammalian cells [15]. Two proteins in T. cruzi, TcRho1 GTPase [26] and TcPRL-1 protein tyrosine phosphatase [27] have so far been shown to be farnesylated.…”

Section: Introductionmentioning

confidence: 99%

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Protein geranylgeranyltransferase-I of Trypanosoma cruzi

Yokoyama

Gillespie

Voorhis

et al. 2008

Molecular and Biochemical Parasitology

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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.

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Protein Prenyltransferases

Hong¹

2004

Handbook of Metalloproteins

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Protein prenyltransferases (PPT) are Zn metalloenzymes that catalyze the covalent attachment of farnesyl or geranylgeranyl isoprenoids to the C‐terminal cysteine residue(s) of many cellular proteins involved in signal transduction and intracellular vesicle transport. There are three subfamilies of protein prenyltransferases: protein farnesyltransferase (PFT), protein geranylgeranyltransferase type I (PGGT‐I), and protein geranylgeranyltransferase type II (PGGT‐II) or Rab geranylgeranyltransferase (RabGGT). All members of PPT are heterodimers composed of an α‐ and a β‐subunit. The crystal structures of PFT, RabGGT, and, in particular, the complex structures of PFT with its substrates or product elucidated the active site configuration and substrate‐binding mode of these enzymes. An intrinsically bound Zn ion was found to be coordinated to an aspartate, a cysteine, and a histidine residue from the β‐subunit. This Zn ion has been shown to be directly involved in the catalysis by activating the protein substrate cysteine thiol for the nucleophilic attack on the C1 atom of the farnesyl or geranylgeranyl diphosphate. PFT has been a primary anticancer drug target, and extensive biochemical and structural studies have been conducted on the kinetics and mechanistic aspects of the enzyme. Progress has also been made in the structural and enzymological studies of RabGGT and PGGT‐I.

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Cloning, Heterologous Expression, and Distinct Substrate Specificity of Protein Farnesyltransferase from Trypanosoma brucei

Cited by 55 publications

References 29 publications

Therapeutic intervention based on protein prenylation and associated modifications

Therapeutic intervention based on protein prenylation and associated modifications

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

Protein Prenyltransferases

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