Characterization of recombinant human farnesyl-protein transferase: Cloning, expression, farnesyl diphosphate binding, and functional homology with yeast prenyl-protein transferases (original) (raw)
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Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases
1991
Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an a/#? heterodimer, comprised of subunits having relative molecular masses of approximately 47 (a) and 46 kDa (#?). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAMlIDPRl (R A M I) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase a-subunit were isolated. Comparison of the amino acid sequences deduced from the a-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase a-subunit. Antisera raised against the RAMl gene product reacted specifically with the #?-subunit of bovine FTase, suggesting that the RAMl gene product is analogous to the bovine FTase #?-subunit. Whereas a raml mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to R A M l , specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-11, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-11. Antisera that specifically recognized the bovine FTase a-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-I1 activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-I1 are comprised of different but homologous #?-subunits and that the a-subunits of FTase and GGTase-I share common features not shared by GGTase-11. Site-specific farnesylation or geranylgeranylation of cellular polypeptides a t a COOH-terminal cysteine residue is a functionally essential post-translational modification (see Ref. 1). Protein acceptor substrates in mammalian cells for farnesylation include the cell-transforming 20-kDa GTPase Ras, nuclear lamin B, and the y-subunit of retinal transducin. Substrates for geranylgeranylation include some 20-kDa
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.
Journal of Biological Chemistry
CAAX motif peptides, which are substrates for isoprenylation, were synthetically derivatized with the light-sensitive benzophenone (Bz) group in order to determine their potential use as catalytic site-directed covalent photocross-linking ligands for one of the enzymes catalyzing protein isoprenylation, farnesyl-protein transferase (FPTase). Bz-peptides could be synthesized with [8H]benzophenone and possessed either one or two benzophenone groups located at or near the peptide's N H z terminus (e.g. the mono-Bz probes Bz-ACVIM and Bz-LPCVVM, and the di-Bz derivatized probe Bz-GY-(Bz)PCVVM, referred to as Bzz-GYPCVVM). Each type of derivatized peptide, behaved as a substrate for farqesylation in vitro without irradiation, while under 366-nm irradiation each demonstrated covalent crosslinking ability as a catalytic site-directed photoaffinity ligand with tissue-purified or enriched but impure fractions from rat and bovine brain FPTase, as well as with a recombinant human FPTase variant, FPTase(pat) expressed in Escherichia coli. Without photoactivation, Bz-ACVIM yielded a & of 37 IIM for the cloned variant of human FPTase. Pseudo first-order photolytic inhibition of FPTase preparations with Bz-peptides, as well as protection from photoinactivation by unmodified -CAAX motif peptides, supported the capacity of these Bz-peptides to serve as co-substrates and their specificity for seeking the catalytic site of the enzyme. SDS-polyacrylamide gel electrophoresis analysis subsequent to photolysis indicated that the mono-Bz-derivatized peptides (e.g.
Biochemistry, 1999
Farnesyl protein transferase (FPT) is an R/ heterodimeric zinc enzyme that catalyzes posttranslational farnesylation of many key cellular regulatory proteins, including oncogenic Ras. On the basis of the recently reported crystal structure of FPT complexed with a CVIM peptide and R-hydroxyfarnesylphosphonic acid, site-directed mutagenesis of the FPT active site was performed so key residues that are responsible for substrate binding and catalysis could be identified. Eight single mutants, including K164NR, Y166FR, Y166AR, Y200FR, H201AR, H248A , Y300F , and Y361F , and a double mutant, H248A /Y300F , were prepared. Steady-state kinetic analysis along with structural evidence indicated that residues Y200R, H201R, H248 , and Y361 are mainly involved in substrate binding. In addition, biochemical results confirm structural observations which show that residue Y166R plays a key role in stabilizing the active site conformation of several FPT residues through cation-π interactions. Two mutants, K164NR and Y300F , have moderately decreased catalytic constants (k cat ). Pre-steady-state kinetic analysis of these mutants from rapid quench experiments showed that the chemical step rate constant was reduced by 41-and 30-fold, respectively. The product-releasing rate for each dropped approximately 10-fold. In pH-dependent kinetic studies, Y300F was observed to have both acidic and basic pK a values shifted 1 log unit from those of the wild-type enzyme, consistent with a possible role for Y300 as an acid-base catalyst. K164NR had a pK a shift from 6.0 to 5.3, which suggests it may function as a general acid. On the basis of these results along with structural evidence, a possible FPT reaction mechanism is proposed with both Y300 and K164R playing key catalytic roles in enhancing the reactivity of the farnesyl diphosphate leaving group. †
Protein Expression and Purification, 1998
Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the ␣and -subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli. Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The -subunit-coding sequence was placed upstream of the ␣-subunit coding sequence linked by overlapping -subunit stop and ␣-subunit start codons. Additionally, the initial 88 codons of the ␣-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E. coli. Since previous attempts at recombinantly expressing FPTase in E. coli from a translationally coupled system have demonstrated that initiation of translation of the ␣subunit is poor, we propose that the optimization of the codons at the start of the ␣-subunit gene leads to the observed high level of recombinant expression.
Inhibitors of farnesyl:protein transferase—A possible cancer chemotherapeutic
1996
The recent interest in inhibitors of farnesyl:protein transferase (FPTase) has resulted in a better understanding of the enzymology of this protein. Rationally designed inhibitors of prenyl transfer have emerged as potential new drug candidates because of the insight gained over bow a prenyl group is enz3'matically transferred onto a peptide thiol. This paper will explore how advances in our understanding of FPTase mediated catalysis has affected the design of FPTase inhibitors as possible cancer therapeutic agents. Without structural information of the enzyme, substrate analogues comprise the first area of drug design: these include peptidomimetics of the four C-terminal amino acids of rasP21 as well as farnesyl diphosphate analogs. In addition, phosphate anion was found to enhance the inhibitory potency of certain compounds known to be competitive with respect to farnesyl diphosphate and therefore incorporation of the phosphate anion may also provide a basis for improved inhibitor design.
Journal of Biological Chemistry, 1998
Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C ؍ cysteine, A ؍ aliphatic residue, X ؍ cysteine, serine, methionine, alanine) and to a lesser degree CAAL. Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine-and leucine-ending substrates both in vitro and in vivo. Substrate overlap of FTase and GGTase I has not been determined to be biologically significant. In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis. Three of the mutations altered the substrate preferences of the wild type enzyme significantly. The ram1p-74 D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type. The ram1p-206 DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates. The ram1p-351 FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL. The ram1p-351 FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate. These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated. The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures. Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes.
Proceedings of the National Academy of Sciences, 1991
The complete amino acid sequence of the a subunit of heterodimeric p2l' protein farnesyltransferase from rat has been deduced from the sequence of a cloned cDNA. The cDNA encodes a 377-amino acid protein that migrates on NaDodSO4/polyacrylamide gels identically to the a subunit purified from rat brain. When introduced into mammalian cells by tansfection, the cDNA for the a subunit produced no immunodetectable protein or farnesyltranderase activity unless the cells were simultaneously transfected with a cDNA encoding j3 subunit. In light of previous evidence that a subunit forms a heterodimer with at least two different 13 subunits, current data suggest a mechanism for coordinating amounts of a and 13 subunits. If an a subunit were stable only as a complex with a P subunit, the number of a subunits would be automatically maintained at a level just sufficient to balance all 13 subunits, thereby avoiding the potentially toxic overaccumulation of free a subunits.
Journal of Biological Chemistry, 1997
Investigation of the comparative activities of various inhibitors of farnesyl:protein transferase (FPTase) has led to the observation that the presence of phosphate or pyrophosphate ions in the assay buffer increases the potency of farnesyl diphosphate (FPP) competitive inhibitors. In addition to exploring the phenomenon of phosphate synergy, we report here the effects of various other ions including sulfate, bicarbonate, and chloride on the inhibitory ability of three FPP competitive compounds: Cbz-His-Tyr-Ser(OBn)TrpNH 2 (2), Cbz-HisTyr-(OPO 4 2؊ )-Ser(OBn)TrpNH 2 (3), and ␣-hydroxyfarnesyl phosphonic acid (4). Detailed kinetic analysis of FPTase inhibition revealed a high degree of synergy for compound 2 and each of these ions. Phosphorylation of 2 to give 3 completely eliminated any ionic synergistic effect. Moreover, these ions have an antagonistic effect on the inhibitory potency of compound 4. The anions in the absence of inhibitor exhibit non-competitive inhibition with respect to FPP. These results suggest that phosphate, pyrophosphate, bicarbonate, sulfate, and chloride ions may be binding at the active site of both free enzyme and product-bound enzyme with normal substrates. These bound complexes increase the potency of FPP competitive inhibitors and mimic an enzyme:product form of the enzyme. None of the anions studied here proved to be synergistic with respect to inhibition of geranylgeranyl transferase I. These findings provide insight into the mechanism of action of FPP competitive inhibitors for FPTase and point to enzymatic differences between FPTase and geranylgeranyl transferase I that may facilitate the design of more potent and specific inhibitors for these therapeutically relevant target enzymes.