Identification and Specificity Profiling of Protein Prenyltransferase Inhibitors Using New Fluorescent Phosphoisoprenoids

Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Journal of the American Chemical Society (Impact Factor: 12.11). 04/2006; 128(9):2822-35. DOI: 10.1021/ja052196e
Source: PubMed


Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.

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    • "Because the CBR is involved in hydrophobic interactions of Rab C-terminal sequences with the hydrophobic patch on REP, we designed Rab7 mutants in which I190 and L192 of the CBR were mutated to polar amino acids (Figure 5A and B). The resulting mutants were purified and subjected to in vitro prenylation with NBD-FPP (Dursina et al, 2006; Wu et al, 2006). As can be seen in Figure 5C, the individual mutations led to a reduction in the rate and total yield of the prenylation reaction. "
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    ABSTRACT: Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely 'outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein-protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that 'learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP.
    Full-text · Article · Sep 2008 · The EMBO Journal
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    ABSTRACT: Modification of proteins with polymers is a viable method to tune protein properties, e.g., to render them more water-soluble by using hydrophilic polymers. We have utilized precision-length, polyethylene glycol-based oligomers carrying a thioester moiety in transthioesterification and native chemical ligation reactions with internal and N-terminal cysteine residues in proteins and peptides. These reactions lead to uniquely modified proteins with an increased solubility in chaotrope- and detergent-free aqueous systems. Polymer modification of internal cysteines is fully reversible and allows generation of stable protein-polymer conjugates for enzymatic manipulations as demonstrated by proteolytic cleavage of a protein construct that was only soluble in buffers incompatible with protease activity before polymer modification. The permanent polymer modification of a Rab protein at its N-terminal cysteine produced a fully active Rab variant that was efficiently prenylated. Thus, PEGylation of prenylated proteins might be a viable route to increase water solubility of such proteins in order to carry out experiments in detergent- and lipid-free systems.
    No preview · Article · Nov 2006 · Bioconjugate Chemistry
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