Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [¹⁴C]-mevalonate, [¹⁴C]isopentenyl- and [¹⁴C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [¹⁴C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg²⁺ ions in combination to the culture of a strain of Arthrobacter gave rise to ¹⁴C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.     

Detection of nonsterol isoprenoids by HPLC-MS/MS

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C₁₈ HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0 μmol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6-10.9 and 4.4-11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway.     

Farnesyl Acetate, a Derivative of an Isoprenoid of the Mevalonate Pathway, Inhibits DNA Replication in Hamster and Human Cells

Cells treated with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme which catalyzes the rate-limiting step of the mevalonate pathway, are arrested prior to the DNA synthesis (S) phase of the cell cycle. Identification of a specific pathway product or products with a role in DNA replication, however, has remained elusive. In this report we demonstrate that farnesyl acetate, a derivative of the key isoprenoid pathway intermediate farnesyl pyrophosphate, inhibits DNA replication in both Chinese hamster ovary cells and human (HeLa) cells. This effect is revealed by measurement of DNA content using fluorescence-activated cell sorter analysis and by measurement of [³H]thymidine incorporation. We show that cells treated with farnesyl acetate retain protein synthesis capacity as DNA replication is inhibited and remain intact as viewed with the vital stain propidium iodide. The inhibition of DNA replication by farnesyl acetate occurs in cells treated with high levels of compactin and in cells lacking HMG-CoA reductase. These results indicate that farnesyl acetate action is not dependent on metabolism through the isoprenoid pathway and is not the result of the loss of a metabolite required for replication nor the accumulation of a metabolite which is inhibitory. In addition, cells treated with farnesyl acetate for over 6 h are irreversibly blocked from progressing through S phase, a phenomenon which differs sharply from the results with compactin, removal of which results in synchronous progression through S phase. Farnesyl acetate also blocks protein prenylation in cells, to a degree comparable to a known farnesylation inhibitor, BZA-5B. We propose that farnesyl acetate is acting in a manner quite different from the metabolic block caused by compactin, causing a rapid and irreversible block of DNA replication.     

Effect of detergents on sterol synthesis in a cell-free system of yeast

In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated. Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined. Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol. Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41). The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture. The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.     

Sites of control of hepatic cholesterol biosynthesis

An inhibition in the conversion of mevalonate to cholesterol has been demonstrated in liver of cholesterol-fed rats by both in vitro and in vivo methods. Synthesis decreased to 30% of the control value after 1 week and 20% after 1 month on a 1% cholesterol diet. After a year, synthesis from mevalonate was almost completely inhibited. The rate of conversion of squalene to cholesterol was not consistently decreased but that of farnesyl pyrophosphate to cholesterol was decreased considerably. The rate of conversion of mevalonate to farnesyl pyrophosphate by a soluble liver enzyme preparation was also decreased in cholesterol-fed animals. Sites of inhibition of cholesterol synthesis were detected before mevalonate, between mevalonate and farnesyl pyrophosphate, and after farnesyl pyrophosphate, probably at the conversion of farnesyl pyrophosphate to squalene. The inhibition of mevalonate conversion to cholesterol developed more slowly than that of acetate and appeared to be secondary to it. The maximum capacities of normal liver homogenates and slices to synthesize cholesterol from mevalonate were shown to be far greater than from acetate. Consequently, sites of inhibition after mevalonate probably do not have a significant effect on the over-all rate of cholesterol synthesis in the intact cholesterol-fed animal.     

Dexamethasone-induced decrease in HMG-CoA reductase and protein-farnesyl transferase activities does not impair ras processing in AR 4-2 J cells

Rat pancreatic acinar cells AR 4-2J respond to dexamethasone by differentiation and a decreased proliferation rate. Protein labelling by [³H]-mevalonolactone, used as a precursor of farnesyl and geranylgeranyl isoprenoid groups, was increased in the presence of dexamethasone. In these same conditions, dexamethasone decreased HMG-CoA reductase activity, leading to a diminished isotopic dilution of the mevalonate precursor. As ras proteins, known to be involved in the regulation of proliferation and differentiation, need to be farnesylated for full biological function, we also measured the level of farnesyl transferase activity and found a dose-dependent decrease in dexamethasone treated cells. Despite these negative effects of dexamethasone on mevalonate pathway, there was no appearance of non-isoprenylated forms of ras, indicating that the level of isoprenoid precursors and farnesyl transferase activity were not limiting in this model.     

A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in eukaryotes.

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). In both mammals and yeast, a non-sterol isoprenoid signal positively regulates the rate of HMGR degradation. To define more precisely the molecule that serves as the source of this signal, we have conducted both pharmacological and genetic manipulations of the mevalonate pathway in yeast. We now demonstrate that farnesyl diphosphate (FPP) is the source of the positive signal for Hmg2p degradation in yeast. This FPP-derived signal does not act by altering the endoplasmic reticulum degradation machinery in general. Rather, the FPP-derived signal specifically modulates Hmg2p stability. In mammalian cells, an FPP-derived molecule also serves as a positive signal for HMGR degradation. Thus, both yeast and mammalian cells employ the same strategy for regulation of HMGR degradation, perhaps by conserved molecular processes.     

Lovastatin induces apoptosis in a metastatic ovarian tumour cell line

Lovastatin is a very specific and potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which regulates a rate-limiting step in the cellular synthesis of isoprenoid and cholesterol. In this study, we demonstrate that treatment of rat ovarian metastatic OV1N cells with lovastatin induces apoptosis. Furthermore, apoptotic death of lovastatin-treated OV1N cells can be prevented by the addition of either mevalonic acid (an immediate metabolite of HMG-CoA) or farnesyl pyrophosphate (one of the downstream products of mevalonic acid metabolism). However, metabolic derivatives of farnesyl pyrophosphate failed to prevent the apoptotic effect of lovastatin on cells. Therefore farnesyl pyrophosphate appears to be important for cell survival and the relationship of this compound to protein farnesylation and apoptosis induction is discussed.     

Prenylation of mammalian Ras protein in Xenopus oocytes.

Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.     

Isoprenoid synthesis during the cell cycle. Studies of 3-hydroxy-3-methylglutaryl-coenzyme A synthase and reductase and isoprenoid labeling in cells synchronized by centrifugal elutriation

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A synthase and reductase were assayed in exponentially growing LM fibroblasts and Friend murine erythroleukemia cells isolated at various stages of the cell cycle by centrifugal elutriation. The activities of these enzymes were similar in all phases of the cell cycle, regardless of whether the cells were cultured in the presence or absence of serum. These observations were confirmed in murine erythroleukemia cells synchronized by recultivation of pure populations of G1 cells. The incorporation of [14C]acetate or 3H2O into sterols decreased by 30-50% in later stages of the cell cycle, whereas the incorporation of [14C]acetate into ubiquinone increased as the cells progressed toward mitosis. Similar changes in the labeling of sterols compared to ubiquinone and dolichol were observed when [3H]mevalonate was used, suggesting that cell cycle-dependent alterations may occur in the flux of farnesyl pyrophosphate into the various branches of the isoprenoid pathway. Synchronized murine erythroleukemia cells incorporated [3H]mevalonate into protein-bound isoprenyl groups at all stages of the cell cycle, and there were no substantial changes in the electrophoretic profiles of these labeled polypeptides. The finding that the activities of the enzymes regulating mevalonate synthesis did not vary substantially during the cell cycle implies that changes in the endogenous mevalonate pool probably do not play a limiting role in regulating cell cycle traverse when cells are undergoing exponential growth. Although small cell cycle-dependent changes may occur in the relative activity of various post-mevalonate branches of the isoprenoid biosynthetic pathway, there is no evidence that synthesis of any major isoprenoid end product is confined exclusively to a specific phase of the cell cycle.     

Phosphorylation of geranyl and farnesyl pyrophosphates by Nm23 proteins/nucleoside diphosphate kinases

The biochemical mechanism(s) by which Nm23 proteins/nucleoside diphosphate kinases suppress tumor metastasis, inhibit cell motility, and affect cellular differentiation are not known. Here we report that Nm23 proteins can phosphorylate geranyl and farnesyl pyrophosphates to give triphosphates. Wild type Nm23-H1 had higher geranyl and farnesyl pyrophosphate kinase activities than did mutants of Nm23-H1 that do not inhibit cell motility. The phosphorylation of farnesyl pyrophosphate appears to occur in vivo as cells with an elevated level of Nm23-H1 contained more farnesyl triphosphate than did control cells. To our knowledge, this is the first report that farnesyl triphosphate exists in cells. The phosphorylation of farnesyl pyrophosphate by Nm23 proteins could alter isoprenoid metabolism, and cells with an elevated level of Nm23 proteins were found to contain more farnesylated 46- and 24-kDa proteins than did control cells. The phosphorylation of geranyl and farnesyl pyrophosphates by Nm23 proteins provides a novel mechanism by which these proteins might exert their biological effects.     

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.     

Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei

The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.     

High-content assay to study protein prenylation

The mevalonate pathway leads to synthesis of cholesterol and isoprenoid lipids. Prenyltransferases attach the isoprenoid lipids to the C-terminus of several small guanosine triphosphate-binding proteins. The prenyl groups are essential for the biological activity of these proteins. The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis, or high serum cholesterol level. Until recently, cellular assays to study protein prenylation have been tedious, low-throughput assays. The authors have developed a high-content imaging-based assay to study protein prenylation. The assay is based on a green fluorescent protein (GFP) reporter, which is tagged with the prenylation motif of human H-Ras. The C-terminus of H-Ras targets GFP to the plasma membrane. When protein prenylation is inhibited, the tagged GFP cannot be localized to plasma membrane but is soluble in the cells. The localization of the GFP reporter can be analyzed in the 96- or 384-well format using automated microscopy and automated image analysis. Information about cell number and nuclear intensity can be obtained from the same images. In compound screening, these readouts provide valuable information about the toxicity of the compounds. The authors have validated their assay using several inhibitors of the mevalonate pathway as well as siRNA against farnesyl pyrophosphate synthase, a critical enzyme in the synthesis of the isoprenoid lipids.     

Indirect stimulation of human Vγ2Vδ2 T cells through alterations in isoprenoid metabolism

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.     

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.     

An oxysterol-derived positive signal for 3-hydroxy- 3-methylglutaryl-CoA reductase degradation in yeast

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In mammals, both a non-sterol isoprenoid signal derived from farnesyl diphosphate (FPP) and a sterol-derived signal appear to act together to positively regulate the rate of HMGR degradation. Although the nature and number of sterol-derived signals are not clear, there is growing evidence that oxysterols can serve in this capacity. In yeast, a similar non-sterol isoprenoid signal generated from FPP acts to positively regulate HMGR degradation, but the existence of any sterol-derived signal has thus far not been revealed. We now demonstrate, through the use of genetic and pharmacological manipulation of oxidosqualene-lanosterol cyclase, that an oxysterol-derived signal positively regulated HMGR degradation in yeast. The oxysterol-derived signal acted by specifically modulating HMGR stability, not endoplasmic reticulum-associated degradation in general. Direct biochemical labeling of mevalonate pathway products confirmed that oxysterols were produced endogenously in yeast and that their levels varied appropriately in response to genetic or pharmacological manipulations that altered HMGR stability. Genetic manipulation of oxidosqualene-lanosterol cyclase did result in the buildup of detectable levels of 24,25-oxidolanosterol by gas chromatography, gas chromatography-mass spectroscopy, and NMR analyses, whereas no detectable amounts were observed in wild-type cells or cells with squalene epoxidase down-regulated. In contrast to mammalian cells, the yeast oxysterol-derived signal was not required for HMGR degradation in yeast. Rather, the function of this second signal was to enhance the ability of the FPP-derived signal to promote HMGR degradation. Thus, although differences do exist, both yeast and mammalian cells employ a similar strategy of multi-input regulation of HMGR degradation.     

The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6+/-0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The K(m) of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6+/-0.5mum; its K(m) for MgATP(2-) was 120+/-7.7mum. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3'-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP(2-). The K(i) for inhibition by geranyl pyrophosphate was 1.3+/-0.2mum; the K(i) for inhibition by farnesyl pyrophosphate was 1.0+/-0.3mum. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.