Context-Dependent Substrate Recognition by Protein Farnesyltransferase † (original) (raw)
Related papers
Journal of Molecular Biology, 2010
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.
Global Identification of Protein Prenyltransferase Substrates
2011
The protein prenyltransferases, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I), catalyze the attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl moiety, respectively, to a cysteine near the C-terminus of a substrate protein targeting it to the membrane. Substrates of the prenyltransferases are involved in a myriad of signaling pathways and processes within the cell, therefore inhibitors targeting FTase and GGTase-I are being developed as therapeutics for treatment of diseases such as cancer, parasitic infection, asthma, and progeria.
Journal of Biological Chemistry, 2012
Background: FTase recognizes and modifies many proteins with C-terminal CA 1 A 2 X sequences. Results: Mutating active site residues Trp-102 and Trp-106 significantly alters FTase peptide selectivity both in vitro and in vivo. Conclusion: FTase substrate selectivity includes negative discrimination that can be relaxed/altered without losing activity. Significance: Deciphering FTase peptide recognition allows creation of bioengineered prenylation pathways and provides a model for other multispecific enzymes. . 3 The abbreviations used are: FTase, protein farnesyltransferase; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; HEPPSO, 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid); dns, dansyl; ORF1, opening reading frame 1 under control of the CMV promoter in the pCAF expression vector; ORF2, opening reading frame 2 under control of the SV40 promoter in the pCAF expression vector.
ChemBioChem, 2014
Prenylation is a post-translational modification wherein an isoprenoid group is attached to a protein substrate by a protein prenyltransferase. Hundreds of peptide sequences are in vitro substrates for protein farnesyltransferase (FTase), but it remains unknown which of these sequences can successfully compete for in vivo prenylation. Translating in vitro studies to predict in vivo protein farnesylation requires determining the minimum reactivity needed for modification by FTase within the cell. Towards this goal, we developed a reporter protein series spanning several orders of magnitude in FTase reactivity as a calibrated sensor for endogenous FTase activity. Our approach provides a minimally invasive method to monitor changes in cellular FTase activity in response to environmental or genetic factors. Determining the reactivity "threshold" for in vivo prenylation will help define the prenylated proteome and identify prenylation-dependent pathways for therapeutic targeting.
Biochemistry, 2020
Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast-and mammalian-based systems have demonstrated FTase
Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions
Journal of Biological Chemistry, 2012
Background: FTase recognizes and modifies many proteins with C-terminal CA 1 A 2 X sequences. Results: Mutating active site residues Trp-102 and Trp-106 significantly alters FTase peptide selectivity both in vitro and in vivo. Conclusion: FTase substrate selectivity includes negative discrimination that can be relaxed/altered without losing activity. Significance: Deciphering FTase peptide recognition allows creation of bioengineered prenylation pathways and provides a model for other multispecific enzymes.
Genome Biology, 2003
Three different protein prenyltransferases (farnesyltransferase and geranylgeranyltransferases I and II) catalyze the attachment of prenyl lipid anchors 15 or 20 carbons long to the carboxyl termini of a variety of eukaryotic proteins. Farnesyltransferase and geranylgeranyltransferase I both recognize a 'Ca 1 a 2 X' motif on their protein substrates; geranylgeranyltransferase II recognizes a different, non-CaaX motif. Each enzyme has two subunits. The genes encoding CaaX protein prenyltransferases are considerably longer than those encoding non-CaaX subunits, as a result of longer introns. Alternative splice forms are predicted to occur, but the extent to which each splice form is translated and the functions of the different resulting isoforms remain to be established. Farnesyltransferase-inhibitor drugs have been developed as anti-cancer agents and may also be able to treat several other diseases. The effects of these inhibitors are complicated, however, by the overlapping substrate specificities of geranylgeranyltransferase I and farnesyltransferase.
Archives of Biochemistry and Biophysics, 1998
Protein farnesyltransferase and protein geranylgeranyltransferase-I catalyze the prenylation of a cysteinyl group located four residues upstream of the carboxyl terminus. The identity of the carboxyterminal residue plays a significant role in determining the ability of compounds to bind to each enzyme and to serve as substrate. We compared the binding and substrate specificities of peptides with carboxyterminal substitutions to determine which residues promote selectivity and which residues promote recognition by both enzymes. Using tetrapeptide inhibitors with the general structure L-penicillamine-valine-isoleucine-X and substrates with the structure Lys-Lys-Ser-Ser-Cys-Val-Ile-X, we measured their respective K i , K m , and k cat values for both recombinant rat protein farnesyltransferase and recombinant rat protein geranylgeranyltransferase-I. We studied the roles of carboxyterminal branched residues (leucine, isoleucine, valine, and penicillamine) and linear residues (methionine, cysteine, homocysteine, alanine, aminobutyrate, and aminohexanoate) in promoting interaction with the enzymes. For protein geranylgeranyltransferase-I, peptide substrates with carboxyterminal branched or linear residues had K m values that were 5to 15-fold greater than the K i values of the corresponding peptide inhibitors. For protein farnesyltransferase, peptide substrates with carboxyterminal branched residues, proline, or homoserine had K m values that were 7-to 200-fold greater than the K i values of the corresponding peptide inhibitors. For protein farnesyltransferase the K m and K i values for peptides ending with linear residues were in general agreement. Our studies indicate that the substrate and inhibitor binding specificities of protein geranylgera-nyltransferase was much more restricted than those of protein farnesyltransferase.
Nonfarnesylated tetrapeptide inhibitors of protein farnesyltransferase
The Journal of biological chemistry, 1991
The protein farnesyltransferase from rat brain was previously shown to be inhibited competitively by tetrapeptides that conform to the consensus Cys-A1-A2-X, where A1 and A2 are aliphatic amino acids and X is methionine, serine, or phenylalanine. In the current studies we use a thin layer chromatography assay to show that most of these tetrapeptides are themselves farnesylated by the purified enzyme. Two classes of tetrapeptides are not farnesylated and therefore act as true inhibitors: 1) those that contain an aromatic residue at the A2 position and 2) those that contain penicillamine (beta,beta-dimethylcysteine) in place of cysteine. The most potent of these pure inhibitors was Cys-Val-Phe-Met, which inhibited farnesyltransferase activity by 50% at less than 0.1 microM. These data indicate that the inclusion of bulky aromatic or methyl residues in a tetrapeptide can abolish prenyl group transfer without blocking binding to the enzyme. This information should be useful in the desig...
Potent and Selective Non-Cysteine-Containing Inhibitors of Protein Farnesyltransferase
Journal of Medicinal Chemistry, 1998
Potent and selective non-thiol-containing inhibitors of protein farnesyltransferase are described. FTI-276 (1) was transformed into pyridyl ether analogue 19. The potency of pyridyl ether 19 was improved by modification of the biphenyl core to that of an o-tolyl substituted biphenyl core to give 29. In addition to 0.4 nM in vitro potency, 29 displayed 350 nM potency in whole cells as the parent carboxylic acid. The o-tolyl biphenyl core dramatically and unexpectedly enhanced the potency of other compounds as exemplified by 46, 47, 48, and 49.