Cloning and expression of a cDNA encoding the alpha subunit of rat p21ras protein farnesyltransferase (original) (raw)
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Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase
Proceedings of the National Academy of Sciences, 1991
We tested 42 tetrapeptides for their ability to bind to the rat brain p2l' protein farnesyltransferase as estimated by their ability to compete with p2lH"in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-Al-A2-X, where positions Al and A2 are occupied by aliphatic amino acids and position X is-occupied by a COOHterminal methionine, serine, or phenylalanine. Charged resi
Purification of ras farnesyl:Protein transferase
Methods, 1990
We describe a method for the purification of farnesyl:protein transferase, an enzyme that transfers a farnesyl group from farnesyl pyrophosphate to a COOH-terminal cysteine in ras proteins, nuclear lamin B, and the 7 subunit of bovine transducin. The enzyme is purified to homogeneity from rat brain cytosol through use of an affinity chromatography step based on the enzyme's ability to specifically bind to a hexapeptide containing the consensus sequence for farnesylation. The purification procedure is reproducible and enables the isolation of microgram amounts of purified enzyme from 50 rat brains. Two methods for assaying enzymatic activity are also described. One assay measures the transfer of [3H]farnesyl from [3H]farnesyl pyrophosphate to recombinant H-ras, and the other measures the transfer of [3H]farnesyl to a biotinylated peptide containing the Cys-AAX COOH-terminal sequence of K-rasB.
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...
Biochemistry, 1993
We have isolated cDNAs encoding the alpha and beta subunits of human farnesyl-protein transferase (FPTase). The proteins encoded by these two cDNAs are 93-95% identical to the corresponding subunits of bovine and rat FPTase and show regions of homology with proteins encoded by Saccharomyces cerevisiae prenyl-protein transferase genes. Human FPTase expressed in Escherichia coli from a translationally coupled operon had kinetic properties similar to those of FPTase isolated from bovine brain. Examination of farnesyl diphosphate binding indicated that while neither individual subunit was capable of isoprenoid binding, a radiolabeled farnesyl diphosphate analog could be specifically photo-cross-linked to the beta subunit of FPTase holoenzyme. To further analyze subunit structure-function and to detect functional similarities with yeast prenyl-protein transferases (FPTase and two geranylgeranyl-protein transferases), amino acid changes homologous to those found in mutant yeast prenyl-protein transferase subunits were made in the subunits of human FPTase. Substitutions in either the alpha or beta subunits that decrease the activity of yeast prenyl-protein transferases were also observed to impair human FPTase. Kinetic analyses showed that these mutant human FPTases have Km and kcat values that are altered with respect to wild-type human FPTase.
N -Arylalkyl Pseudopeptide Inhibitors of Farnesyltransferase
Journal of Medicinal Chemistry, 1998
a nd, not determined. b Concentration of compound required to inhibit 50% of FPTase-catalyzed incorporation of [ 3 H]FPP into recombinant human Ha-Ras protein by 50%. Those values marked with + were obtained using enzyme from bovine brain at a concentration of approximately 1 nM, and those marked with * used 10 pM enzyme concentration. c Inhibition of bovine type-I geranylgeranyltransferase. 8 Assay results are reported as concentration ( SEM for the number of determinations shown in parentheses. With one determination, the values are estimated to be reliable within 2-fold.
Context-Dependent Substrate Recognition by Protein Farnesyltransferase †
Biochemistry, 2009
Prenylation is a posttranslational modification whereby C-terminal lipidation leads to protein localization to membranes. A C-terminal "Ca 1 a 2 X" sequence has been proposed as the recognition motif for two prenylation enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I. To define the parameters involved in recognition of the a 2 residue, we performed structure-activity analysis which indicates that FTase discriminates between peptide substrates based on both the hydrophobicity and steric volume of the side chain at the a 2 position. For nonpolar side chains, the dependence of the reactivity on side chain volume at this position forms a pyramidal pattern with a maximal activity near the steric volume of valine. This discrimination occurs at a step in the kinetic mechanism that is at or before the farnesylation step. Furthermore, a 2 selectivity is also affected by the identity of the adjacent X residue, leading to context-dependent substrate recognition. Context-dependent a 2 selectivity suggests that FTase recognizes the sequence downstream of the conserved cysteine as a set of two or three cooperative, interconnected recognition elements as opposed to three independent amino acids. These findings expand the pool of proposed FTase substrates in cells. A better understanding of the molecular recognition of substrates performed by FTase will aid in both designing new FTase inhibitors as therapeutic agents and characterizing proteins involved in prenylation-dependent cellular pathways.
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.
The Journal of biological chemistry, 1992
The separate catalytic roles of Zn2+ and Mg2+ and the specificity of the prenyl pyrophosphate-binding site of the rat brain protein farnesyltransferase were explored using a purified enzyme preparation. The binding of p21Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl2, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21Hras required Mg2+. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21Hras. It also was not transferred to a modified p21Hras containing COOH-terminal leucine, a protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the protein farnes...
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.