Showing papers on "Farnesyl pyrophosphate published in 2000"
TL;DR: By inhibiting expression of PAI-1 from SMCs and ECs while increasing expression of tPA from ECs, simvastatin may alter the local fibrinolytic balance within the vessel wall toward increased fibralelytic capacity that, in turn, would reduce thrombotic risk after plaque rupture.
Abstract: The clinical benefit of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may derive from a qualitative, functional change in atherosclerotic lesions in addition to their lipid-lowering properties. We examined whether statins altered expression of the major determinants of fibrinolytic balance, plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (tPA) in human vascular smooth muscle (SMC) and endothelial (EC) cells. Simvastatin reduced levels of PAI-1 antigen released from SMCs and ECs stimulated with platelet-derived growth factor or transforming growth factor-beta (IC(50) approximately 1 micromol/L). Levels of EC-derived tPA increased 2-fold over the same concentrations of simvastatin that inhibited release of PAI-1. Simvastatin's inhibitory effect was mimicked by C3 exoenzyme and prevented by geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate, suggesting the involvement of geranylgeranyl-modified intermediates. Decreased PAI-1 antigen was correlated with reduced mRNA transcription and activity of the PAI-1 promoter. By inhibiting expression of PAI-1 from SMCs and ECs while increasing expression of tPA from ECs, simvastatin may alter the local fibrinolytic balance within the vessel wall toward increased fibrinolytic capacity that, in turn, would reduce thrombotic risk after plaque rupture.
270 citations
TL;DR: Data suggest that FTase catalyzes protein farnesylation by an associative mechanism with an "exploded" transition state where the metal-bound peptide/protein sulfur has a partial negative charge, the C1 of FPP has apartial positive charge, and the bridge oxygen between C1 and the alpha phosphate of F PP has a Partial negative charge.
Abstract: Protein farnesyltransferase is a zinc metalloenzyme that catalyzes the transfer of a 15-carbon farnesyl group to a conserved cysteine residue of a protein substrate. Both electrophilic and nucleophilic mechanisms have been proposed for this enzyme. In this work, we investigate the detailed catalytic mechanism of mammalian protein farnesyltransferase by measuring the effect of metal substitution and/or substrate alterations on the rate constant of the chemical step. Substitution of cadmium for the active site zinc enhances peptide affinity approximately 5-fold and decreases the rate constant for the formation of the thioether product approximately 6-fold, indicating changes in the metal-thiolate coordination in the catalytic transition state. In addition, the observed rate constant for product formation decreases for C3 fluoromethyl farnesyl pyrophosphate substrates, paralleling the number of fluorines at the C3 methyl position and indicating that a rate-contributing transition state has carbocation character. Magnesium ions do not affect the affinity of either the peptide or the isoprenoid substrate but specifically enhance the observed rate constant for product formation 700-fold, suggesting that magnesium coordinates and activates the diphosphate leaving group. These data suggest that FTase catalyzes protein farnesylation by an associative mechanism with an "exploded" transition state where the metal-bound peptide/protein sulfur has a partial negative charge, the C1 of FPP has a partial positive charge, and the bridge oxygen between C1 and the alpha phosphate of FPP has a partial negative charge. This proposed transition state suggests that stabilization of the developing charge on the carbocation and pyrophosphate oxygens is an important catalytic feature.
68 citations
TL;DR: The effect on the GF-induced activation of intermediates in signal transduction pathways was investigated in order to gain insight into the mechanism of action within the cells, and Rho seems to be a good candidate as a target for inhibitors of PGGT-1.
64 citations
Journal Article•
TL;DR: The clinical benefit of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may derive from a qualitative, functional change in atherosclerotic lesions in addition to their lipid-lowering properties.
Abstract: —The clinical benefit of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may derive from a qualitative, functional change in atherosclerotic lesions in addition to their lipid-lowering properties. We examined whether statins altered expression of the major determinants of fibrinolytic balance, plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (tPA) in human vascular smooth muscle (SMC) and endothelial (EC) cells. Simvastatin reduced levels of PAI-1 antigen released from SMCs and ECs stimulated with platelet-derived growth factor or transforming growth factor-β (IC50 ≈1 μmol/L). Levels of EC-derived tPA increased 2-fold over the same concentrations of simvastatin that inhibited release of PAI-1. Simvastatin’s inhibitory effect was mimicked by C3 exoenzyme and prevented by geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate, suggesting the involvement of geranylgeranyl-modified intermediates. Decreased PAI-1 antigen was correlated with ...
62 citations
TL;DR: The results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement.
Abstract: Farnesyl pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate, a key intermediate in sterol and sesquiterpene biosynthesis. Using a polymerase chain reaction-based approach, we have characterized LeFPS1, a tomato (Lycoperscion esculentum cv Wva 106) fruit cDNA, which encodes a functional FPS. We demonstrate that tomato FPSs are encoded by a small multigenic family with genes located on chromosomes 10 and 12. Consistent with farnesyl pyrophosphate requirement in sterol biosynthesis, FPS genes are ubiquitously expressed in tomato plants. Using an LeFPS1 specific probe, we show that the corresponding gene can account for most of FPS mRNA in most plant organs, but not during young seedling development, indicating a differential regulation of FPS genes in tomato. FPS gene expression is also under strict developmental control: FPS mRNA was mainly abundant in young organs and decreased as organs matured with the exception of fruits that presented a biphasic accumulation pattern. In this latter case in situ hybridization studies have shown that FPS mRNA is similarly abundant in all tissues of young fruit. Taken together our results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement.
60 citations
TL;DR: The kinetics and mechanism of this reaction pathway using Escherichia coli UPPs showed that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton, which indicated a step after product generation was rate limiting.
Abstract: Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate. We investigated the kinetics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were found. In the presence of excess FPP, the intermediates (C(25)-C(50)) accumulated. Under a condition with enzyme and FPP in excess of IPP, instead of C(20)-geranylgeranyl pyrophosphate, C(20), C(25), and C(30) were the major products. The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Triton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k(cat) value matched the rate constant of each IPP condensation obtained from the enzyme single-turnover experiments. This suggested that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton. In the absence of Triton, the intermediates formed and disappeared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting. This was further supported by a burst product formation. Judging from the accumulation level of C(55), C(60), and C(65), their dissociation from the enzyme cannot be too slow and an even slower enzyme conformational change with a rate of 0.001 s(-1) might govern the UPPs reaction rate under the steady-state condition in the absence of Triton.
49 citations
TL;DR: In this paper, trans,trans-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-fernesene, was partially purified from skin tissue of 'Delicious' apples and characterized.
Abstract: ADDITIONAL INDEX WORDS. Malus ×domestica , storage, conjugated triene alcohol (CTOL), farnesol, methyl heptenone (MHO), methyl heptenol (MHOL) ABSTRACT. To decipher the relation between α-farnesene metabolism and the development of superficial scald in apples, trans,trans-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-farnesene, was partially purified from skin tissue of 'Delicious' apples ( Malus ×domestica Borkh.) and characterized. Total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. α-Farnesene synthase was purified 70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as Mg 2+ or Mn 2+ for activity. It exhibited allosteric kinetics, S (0.5) for farnesyl pyrophosphate being 84 ± 18 μmol·L -1 , and a Hill coefficient (n H) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 °C, while 50% of the maximal activity was retained at 0 °C. In vivo α-farnesene synthase activity was minimal at harvest, then increased rapidly during 16 weeks storage in air at 0 °C, and decreased during further storage. Activity of α-farnesene synthase, α-farnesene content, and conjugated triene alcohol (the putative scald- causing oxidation product of α-farnesene) content in skin tissue were not correlated to the inherent nature of scald susceptibility or resistance in 11 apple cultivars tested.
48 citations
TL;DR: AGPP is the first example of a class of compounds that are alternate substrates for protein isoprenylation that are not inhibitors of squalene synthase, and the first efficiently transferable analogue of FPP to be modified at the omega-terminus that provides a platform for additional analogues to probe the biological function of protein farnesylation.
Abstract: The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its membrane localization and is required for both its biological activity and ability to induce malignant transformation. We describe the design and synthesis of a farnesyl pyrophosphate (FPP) analogue, 8-anilinogeranyl pyrophosphate 3 (AGPP), in which the ω-terminal isoprene unit of the farnesyl group has been replaced with an aniline functionality. The key steps in the synthesis are the reductive amination of the α,β-unsaturated aldehyde 5 to form the lipid analogue 6, and the subsequent conversion of the allylic alcohol 7 to the chloride 8 via Ph3PCl2 followed by displacement with [(n-Bu)4N]3HP2O7 to give AGPP (3). AGPP is a substrate for protein farnesyltransferase (FTase) and is transferred to Ras by FTase with the same kinetics as the natural substrate, FPP. AGPP is highly selective, showing little inhibitory activity against either geranylgeranyl-protein transferase type I (GGTase I) (Ki = 0.06 μM, IC50 = 2...
46 citations
TL;DR: It is demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM), and the implications of the low level of discrimination by GGT enzyme for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.
Abstract: Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rab family by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated to what extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp) and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoids indicated that in all cases association is rapid, with rate constants in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak binding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K(d) of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate constant of 0.082 s(-1) which is very similar to that of GGpp. The implications of the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.
36 citations
TL;DR: Results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding and the E213 residue in region V is also important in IPPbinding as well as catalysis.
Abstract: Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs Its IPP K(m) value has only minor change The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat) The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding The E213 residue in region V is also important in IPP binding as well as catalysis Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs
31 citations
TL;DR: The phosphorylation of geranyl and farnesyl pyrophosphates by Nm23 proteins provides a novel mechanism by which these proteins might exert their biological effects, and this is the first report thatfarnesyl triphosphate exists in cells.
TL;DR: A pepper 5-epi-aristolochene synthase gene family of 5–8 members was demonstrated by Southern blot and the nucleotide sequence of the proposed gPEAS1 promoter showed putative stimuli- and tissue-specific responsive elements.
Abstract: Phytoalexins play an important role in the inducible defense responses of plants against diseases caused by fungi. Some enzymes, involved in the respective biosynthetic pathways, catalyze key steps. Farnesyl pyrophosphate (FPP) is a key intermediate in the biosynthesis of terpenes. It may be converted into several cyclic and some acyclic sesquiterpenic derivatives, sterol precursors, or to geranylgeranyl pyrophosphate according to the requirements of the plant cells. Specific cyclization reactions of FPP are catalyzed by particular sesquiterpene cyclases. The 5-epi-aristolochene synthase (EAS) enzyme of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) produce the 5-epi-aristolochene, which is the immediate precursor of the bicyclic phytoalexin capsidiol. Oligonucleotides homologous to 3′-end specific regions of tobacco and Hyoscyamus muticus inducible sesquiterpene cyclase genes were used with the system for rapid amplification of cDNA ends (3′-RACE) technique to prepare pepper EAS-cDNA fragments (PEAS-cDNA). Three specific PEAS-cDNAs were isolated (PEAS1, PEAS18, and PEAS55). Northern blots of total RNA samples from pepper plant tissues challenged with cellulase or Phytophthora capsici showed different expression levels of the respective transcripts. PEAS1 was used to identify the corresponding elicitor-stimulated gene (gPEAS1). The nucleotide sequence of the proposed gPEAS1 promoter showed putative stimuli- and tissue-specific responsive elements. A pepper 5-epi-aristolochene synthase gene family of 5–8 members was demonstrated by Southern blot.
TL;DR: A-176120 is a potent FPP mimetic with both antitumour and anti-angiogenic properties and had an additive effect in combination with cyclophosphamide in nude mice inoculated with K-ras NIH3T3 transformed cells.
TL;DR: Incubation of epicubenol synthase with farnesyl pyrophosphate in the presence of 11.1 atom% H2(18)O gave epicubenols in which the hydroxyl oxygen atom was shown to be derived exclusively from water, as established by GC-selected ion monitoring MS of the derived TMS-epicubenol derivative.
Journal Article•
TL;DR: A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyl transferase, which may be useful in blocking the action of Ras onco-proteins.
Abstract: The 21-kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways. GTP activated Ras interacts directly with the Raf protein to recruit the MAP kinases and their subordinates. Attachment of Ras protein to the plasma membrane that requires farnesylation by farnesyl pyrophosphate at its C-terminus, is essential for its biological activity. Ras oncogenes are associated with a wide variety of solid tumors and leukemias for which existing chemotherapeutics have limited utility. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyl transferase. These inhibitors may be useful in blocking the action of Ras onco-proteins.
TL;DR: The farnesyl transferase inhibitors were observed to slow the time course of oocyte maturation but did not block the IGF-1-induced maturation response.
Abstract: Preincubation of Xenopus laevis oocytes with insulin or insulin-like growth factor 1 (IGF-1) resulted in inhibition of farnesyl transferase (FTase) activity measured both in vivo (after microinjection of tritiated farnesyl pyrophosphate and Ras-CVIM into oocytes) and in extracts using a filtration assay. FTase activity measured in oocyte extracts was inhibited 55% after a 20 min treatment of oocytes with 1 microM insulin or 10 nM IGF-1. The apparent IC(50) for inhibition of oocyte FTase by IGF-1 is 0.3 nM. The observed decrease in FTase activity was apparently not due to translocation of enzyme from cytosol to membrane, since activities measured both in soluble extracts and resuspended crude pellets displayed comparable levels of inhibition following hormone treatment. Using a hexapeptide (TKCVIM) as substrate, FTase activity was also inhibited 65% when oocytes were pretreated with 10 nM IGF-1. Two FTase inhibitors [(alpha-hydroxyfarnesyl) phosphonic acid (HFPA) and chaetomellic acid A (CA)] effectively inhibited Xenopus oocyte FTase by 80-90% when added to assay mixtures (IC(50) values of 338 +/- 96 nM HFPA and 232 +/- 80 nM CA) or after incubation of oocytes in drug before preparation of soluble extracts for assay (IC(50) values of 7 +/- 6 nM HFPA and 328 +/- 128 nM CA). The farnesyl transferase inhibitors were observed to slow the time course of oocyte maturation but did not block the IGF-1-induced maturation response. J. Exp. Zool. 286:193-203, 2000.
TL;DR: Experimental studies, using animal cancer models, have demonstrated that some monoterpenes possess anticarcinogenic properties, acting at different cellular and molecular levels, and it seems clear that monoterspenes could be considered as effective, nontoxic dietary antitumorigenic agents that hold promise as a novel class of anticancer drugs.
Abstract: Monoterpenes are compounds found in the essential oils extracted from many plants, including fruits, vegetables, spices and herbs These compounds contribute to the flavor and aroma of plant from which they are extracted Monoterpenes are acyclic, monocyclic, or bicyclic C30 compounds synthesized by monoterpene synthases using geranyl pyrophosphate (GPP) as substrate GPP is also the precursor in the synthesis of farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), two important compounds in cell metabolism of animals, plants and yeast Monoterpene cyclases produce cyclic monoterpenes through a multistep mechanism involving a universal intermediate, a terpinyl cation which can be transformed to several compounds Experimental studies, using animal cancer models, have demonstrated that some monoterpenes possess anticarcinogenic properties, acting at different cellular and molecular levels From these discoveries it seems clear that monoterpenes could be considered as effective, nontoxic dietary antitumorigenic agents that hold promise as a novel class of anticancer drugs
01 Jan 2000
TL;DR: Sesquiterpenes are the C15 representatives of the terpenoid family of natural products and diverge from higher isoprenoid biosynthesis at the level of farnesyl pyrophosphate (Ruzicka 1953; Cane 1981, 1990; Glasby 1982; Beale 1990a,b, 1991; Fraga 1991, 1997; Conolly and Hill 1992; Buckingham 1994; Dewick 1995, 1997).
Abstract: As already discussed in Volume 60, sesquiterpenes are the C15 representatives of the terpenoid family of natural products and they diverge from higher isoprenoid biosynthesis at the level of farnesyl pyrophosphate (Ruzicka 1953; Cane 1981, 1990; Glasby 1982; Beale 1990a,b, 1991; Fraga 1991, 1997; Conolly and Hill 1992; Buckingham 1994; Dewick 1995, 1997).
01 Mar 2000
TL;DR: The chemotherapeutic agent that illustrated the most promising synergy with other drugs was taxol, and the combination of taxol with the HMG-CoA reductase inhibitor, mevastatin, and with the differentiating agent, phenylbutyrate showed the most significant synergy.
Abstract: : After reviewing all of the drug combinations tested, the chemotherapeutic agent that illustrated the most promising synergy with other drugs was taxol. In particular, the combination of taxol with the HMG-CoA reductase inhibitor, mevastatin, and with the differentiating agent, phenylbutyrate, showed the most significant synergy. However, the combination of taxol with combination of mevastatin and phenylbutyrate showed no increase in antitumor efficacy compared with taxol plus mevastatin or taxol plus phenylbutyrate. These results make sense based on what is known about the mechanism of action of taxol, mevastatin, and phenylbutyrate. Ras, rho and related proteins play a critical role in signal transduction events regulating cell growth and motility. These proteins typically must undergo post translational modification involving the addition of either farnesol- or geranylgeranyl- groups. This reaction involves a condensation reaction between the protein and farnesol pyrophosphate or geranylgeranyl pyrophosphate. In this reaction, the pyrophosphate acts as a leaving group and resulting addition of farnesol- or genanylgeranyl to a cysteine on the protein. The farnesol- and geranylgeranyl groups are composed of isoprenyl- subunits. These isoprenyl subunits are synthesized from acetic acid by a pathway that starts with HMG-CoA reductase. Subsequent steps in the pathway include the formation of mevalonate, mevalonate 5-pyrophosphate and isopentenyl pyrophosphate. In addition to the formation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, isopentenyl pyrophosphate is an intermediate in the synthesis of a wide range of important molecules, including cholesterol, steroid hormones, and coenzyme QlO. Mevastatin, phenylbutyrate and taxol all inhibit separate steps along the pathway leading from acetate to the famesylation or geranylgeranylation of ras-like proteins.