We present experimental evidence for high-ranked human proteins predicted to be affected by anticancer drugs inhibiting prenylation. Introduction Protein prenylation is facilitated by three eukaryotic enzymes with partially overlapping substrate specificities [1C3]. targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domainCcontaining FLJ32421 (termed BROFTI), and Rab28 (short isoform) as unique farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is usually presented for the selective farnesylation of targets with an evolutionary conserved modification site. Author Summary Various cellular functions require reversible membrane localization of proteins. This is often facilitated by attaching lipids to the respective proteins, thus anchoring them to the membrane. For example, addition of prenyl lipid anchors (prenylation) is usually directed by a motif in the protein sequence that can be predicted using a recently developed method. We describe the prediction of protein prenylation in all currently known proteins. The annotated results are available as an online database: PRENbase. A ranking of the predictions is usually introduced, assuming that existence of a prenylation sequence motif in related proteins from different species (evolutionary conservation) relates to functional importance of the lipid anchor. We present experimental evidence for high-ranked human proteins predicted to be affected by anticancer drugs inhibiting prenylation. Introduction Protein prenylation is usually facilitated by three eukaryotic enzymes with partially overlapping substrate specificities [1C3]. Farnesyltransferase (FT) and geranylgeranyltransferase I (GGT1) recognize the so-called C-terminal CaaX box of substrate proteins to attach either a farnesyl (15 carbons) or geranylgeranyl (20 carbons) anchor to the conserved cysteine via a thioether linkage. Rab geranylgeranyltransferase or geranylgeranyltransferase II (GGT2) requires the formation of a complex of the substrate protein with E-7386 a dedicated escort protein, REP (Rab escort protein) [4], and typically attaches two geranylgeranyl anchors to C-terminal cysteines in motifs such as -XXXCC, -XXCXC, -XXCCX, -XCCXX, or -CCXXX [5]. Isoprenyl lipid anchor attachment to C-termini of proteins not only serves for membrane targeting but can also be crucial for proteinCprotein interactions [6]. Inhibition of protein prenylation is a promising approach for developing anti-cancer drugs [7] E-7386 as well as for treating parasitic diseases [8,9]. Therefore, it is of great scientific and applied medical interest to clarify which proteins and pathways are affected by farnesyl- or geranylgeranyltransferase inhibitors in human cells or in unicellular parasites. Based on the refinement of descriptions of sequence motifs recognized by the three enzymes (FT, GGT1, and GGT2) in substrate proteins, we have recently developed amino acid sequenceCbased predictors for various types of protein prenylation (PrePS [10]). PrePS is available as a WWW support (http://mendel.imp.ac.at/sat/PrePS/index2.html). Since the rate of false-positive predictions of PrePS is usually low (for proteins with CXXX C-terminus, the false-positive rate is usually estimated at 5% at a sensitivity for true targets of 98% [10]), this tool is appropriate for large-scale automated annotation (for example, for proteome scans). E-7386 In this work, we apply PrePS to finding all potential protein substrates of the three prenyltransferases. With the analyses of these protein sets, it can be decided which prenylation targets E-7386 Rabbit Polyclonal to OR2L5 are preferentially affected if enzyme-specific prenyltransferase inhibitors are applied. As previous experience with a similar project (the application of the MyrPS/NMT myristoylation predictor [11,12] for searching the nonredundant database and the resulting MYRbase [13]) has shown,.