Mapping the isoprenoid binding pocket of PDEdelta by a semisynthetic, photoactivatable N-Ras lipoprotein.

Biologically functional Ras isoforms undergo post-translational modifications starting with farnesylation of the most C-terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatiotemporal distribution. The delta subunit of phosphodiesterase (PDEdelta) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDEdelta can be mapped by coupling with a semisynthetic N-Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included parts of beta-sheet 9 of PDEdelta, which contains the highly conserved amino acids V145 and L147. Modeling of the PDEdelta-geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDEdelta.

Prenylation of Ras facilitates hSOS1-promoted nucleotide exchange, upon Ras binding to the regulatory site.

The oncoprotein Ras is anchored in lipid membranes due to its C-terminal lipid modification. The ubiquitously expressed Ras nucleotide exchange-factor hSOS1 promotes nucleotide exchange and thus Ras activation. This reaction is enhanced by a positive feedback loop whereby activated Ras binds to an allosteric site of SOS to enhance GEF activity. Here we present biochemical data showing that prenylation of both active site bound and allosterically bound N-Ras is required for efficient hSOS1-promoted nucleotide exchange. Our results indicate that prenyl sensitivity of the allosteric feedback-activation is mediated by the PH domain of hSOS1. Farnesylation of Ras thereby allows hSOS1 to bind even GDP-loaded allosteric regulator to maintain basal hSOS1-activity.

Synthesis and biological activity of photoactivatable N-ras peptides and proteins.

A modular strategy for the assembly of farnesylated N-Ras heptapeptides carrying a photoactivatable benzophenone (BP) group within the lipid residue is described. This strategy is based on the fragment condensation of a N-terminal hexapeptide synthesized on the solid support with a cysteine methyl ester which is modified with different farnesyl analogues, incorporating the photophor. At the N-terminus of the peptides different functional groups can be attached, e.g., biotin for product enrichment and detection after photoactivation or a maleimido (MIC) linker, allowing for the coupling to proteins carrying a C-terminal free cysteine. Using this strategy, 24 peptides were synthesized, incorporating farnesyl analogues with four different chain lengths. Two of these photoactivatable conjugates were ligated to oncogenic human N-RasG12V Delta 181. A cellular transformation assay revealed that the semisynthetic proteins retain their biological activity despite the photolabel. The first photolabeling experiments with a geranyl-BP-labeled N-Ras construct and the farnesyl-sensitive guanine nucleotide exchange factor hSos1 indicate that this photoaffinity labeling system can be particularly useful for studying protein-protein interactions, e.g., the participation of the farnesyl group in Ras signaling, which is still discussed with controversy.

Identification of mono- and bisubstrate inhibitors of protein farnesyltransferase and inducers of apoptosis from a pepticinnamin E library.

A library of 51 analogues of the naturally occurring protein farnesyltransferase inhibitor pepticinnamin E was investigated biologically. Several compounds with pronounced inhibitory activity were discovered with the lowest IC(50) value reaching 1 microM. The library contains inhibitors which are competitive to either farnesylpyrophosphate or the peptide substrate and a bisubstrate inhibitor. This activity is supported and rationalized by molecular modelling experiments and different binding modes of the inhibitors deduced from them. Several compounds induced apoptosis in a Ras-transformed tumour cell line, and in one case this correlated with farnesyltransferase-inhibiting activity.