Evaluation of protein farnesyltransferase substrate specificity using synthetic peptide libraries

Abstract

Farnesylation, catalyzed by protein farnesyltransferase (FTase), is an important posttranslational modification guiding cellular localization. Recently predictive models for identifying FTase substrates have been reported. Here we evaluate these models through screening of dansylatedGCaaS peptides, which also provides new insights into the protein substrate selectivity of FTase.Farnesylation is a posttranslation modification that tags proteins with a farnesyl (C 15 ) isoprenoid supplied by farnesyl diphosphate (FPP) for the purpose of locating the protein to cellular membranes. These lipidated protein substrates are modified by farnesyltransferase (FTase) on their C-terminal FTase recognition sequence called the CaaX box. When it was found that farnesylation occurs on oncogenic Ras proteins 1 , FTase became a chemotherapeutic drug target. 2 Although initially developed based on a simple paradigm where FTIs would target Ras-driven tumors, FTIs have proven to work via a complex mechanism, and their activity is now attributed to the perturbation of a number of cellular proteins. 2, 3The complex and unexpected biology observed with FTIs has made a precise definition of the set of farnesylated proteins in a human cell critically important. It is not known how many proteins in the cell are farnesylated or what are the critical targets of FTIs. Early biochemical studies of Brown and Goldstein 4 and the Merck group 5 demonstrated that tetrapeptides bearing a cysteine, two amino acids, and the appropriate X residue are farnesylated and serve as the minimum substrate for FTase recognition. Recent modelling studies have provided predictions for FTase Ca 1 a 2 X box specificity and thus its protein substrates. 6, 7 These models are only predictive and require additional investigation 8, 9 to determine cellular protein farnesylation. Using traditional biological tools (radiolabeling and/or western blot analysis), it would be timeconsuming to confirm the cellular farnesylation of these hypothetical FTase substrates. Therefore, a screening approach to validate that FTase accepts and modifies the minimal substrate Ca 1 a 2 X boxes of a select group of these proteins would be useful ( Figure 1).As part of our laboratory's investigation into FTase specificity, we have synthesized a library of Dansyl-GCa 1 a 2 S pentapeptides representing FTase substrate candidates. The sequences were identified from a Swissprot database search for carboxyl-terminal Ca 1 a 2 S boxes. Sequences were chosen to represent a) biologically important farnesylated proteins, and b) † These authors contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the...

Context-Dependent Substrate Recognition by Protein Farnesyltransferase

“…For all of these peptide panels, the reactivity of FTase with the peptides decreases with increasing polarity of the a 2 residue for the polar amino acids as defined in Figure 2a; in fact, the reactivity of FTase with some of the peptides with charged a 2 residues is below the detection limit of the fluorescence assay (Table 1). This trend indicates that the preference for hydrophobic residues at the a 2 position is general, as previously proposed (1, 3, 14, 17, 25, 44, 45). In contrast, for the “nonpolar” amino acids, the dependence of reactivity on the volume of the a 2 residue is dependent on the identity of the X group (Figure 3); proline-containing substrates (open triangles) are excluded from the correlation analysis as they appear to be outlier points, possibly due to changes in conformation of the peptide substrate.…”

Synthesis and screening of a CaaL peptide library versus FTase reveals a surprising number of substrates

“…In both cases, the isoprenoid is attached to the cysteine in a C-terminal “Ca 1 a 2 X” sequence. While the “a” residues guide substrate ability,3, 4 the X position is the most important selectivity factor for FTase versus GGTase-I. Those peptides bearing X residues of Ser, Gln, and Met are farnesylated (Ras proteins and others) and those bearing Leu are geranylgeranylated (Rho proteins and others).…”

“…It is not known if the alternate farnesylation of any geranylgeranylated proteins would confer resistance to GGTIs, and this is a key question with regard to their evaluation 10. As a complement to proteomic methods,13, 14 we have employed peptide libraries to evaluate FTase substrate specificity 3. In this study, we screened 41 dansyl-GCaaL (DnGCaaL) peptides versus FTase, and found a surprising number of efficient substrates.…”

“…A continuous spectrofluorimetric assay was used to determine FTase substrate activity for the DnGCaaL peptides 3, 15. The FTase substrate DnGCVLS, representing the CaaX box of H-Ras, was used as the control.…”

Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

“…Prenylation is required for the proper function of many proteins, including members of the Ras and Rho superfamilies of small GTPases (2,6). FTase is known to modify a large number of proteins within the cell (7)(8)(9)(10); recent experimental and theoretical/computational studies using small peptide substrates suggest that several hundred proteins within the human proteome may be farnesylated (11)(12)(13)(14). Based on peptide reactivity and structural studies of FTase-substrate complexes, a minimal substrate recognition motif for FTase is a peptide or protein containing a cysteine four amino acids from the C terminus (-CXXX).…”