Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Abstract

Prenylation is a post-translational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development and farnesyltransferase inhibitors (FTIs) are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for FTI efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under singleturnover conditions. Based on these results, a second library was designed that yielded an additional 29 novel multiple-turnover FTase substrates and 45 single-turnover substrates. The two classes of substrates exhibit different specificity requirements. Efficient multiple-turnover reactivity correlates with the presence of a nonpolar amino acid at the a 2 position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca 1 a 2 X sequence model. In contrast, the sequences of the single-turnover substrates vary significantly more at both the a 2 and X residues and are not well-described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of single-versus multipleturnover peptide reactivity with FTase.

Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

“…25 That sparse sampling of the total sequence space available from varying three residues (8000 possibilities) was quite useful for understanding prenylation specificity in the context of human biochemistry. However, such a study cannot reveal the complete specifity profile of the enzyme given the limited sampling.…”

Protein farnesylation inhibitors cause donut-shaped cell nuclei attributable to a centrosome separation defect

“…Despite well-documented antiproliferative and proapoptotic effects of FTIs on cancer cells (24), their mode of antitumor activity has remained elusive, in part because of the large number (>100) of protein substrates for protein farnesyltransferase (25,26). Induction of apoptosis and loss of bipolar spindle formation with arrest of cells in G2-M phase with a rosette-like chromatin distribution have been reported previously in the setting of FTI treatment (24,27,28), without any mechanism.…”

Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

“…Structural Determinants of CAAX Substrate Selection-The identity of the a 2 and X residues determines Ca 1 a 2 X (cysteine, two generally aliphatic residues, and X specificity-determining variable residue; X residue, the specificity determining residue of the CAAX motif) recognition and specificity (36,55). Some of the most potent FTIs are substrate mimetics, including L-744,832, which exhibits mild antifungal activity against C. neoformans in our disc diffusion and MIC assays.…”