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PubMed1998

3-Hydroxy-3-methyl-glutaryl-CoA reductase in Trypanosoma (Schizotrypanum) cruzi: subcellular localization and kinetic properties.

The subcellular localization of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes the first committed step of the mevalonate pathway, was investigated in Trypanosoma cruzi epimastigotes using well-established cell fractionation procedures. It was found that ca. 80% of the activity of the enzyme was associated with the glycosomes, microbody-like organelles unique to kinetoplastid protozoa which contain most of the enzymes of the glycolytic pathway, while the rest of the activity was found in the soluble (cytoplasmatic) fraction, with almost no activity associated with microsomes. The glycosome-associated enzyme is not membrane-bound as it was recovered quantitatively in the aqueous phase of the biphasic system formed by Triton X-114 at 30 degrees C. Studies with digitonin-permeabilized intact epimastigotes demonstrated the presence of two pools of soluble HMG-CoA reductase in these cells, associated to the cytoplasmic and glycosomal compartments. Steady-state kinetic studies of the glycosome-associated enzyme indicated classical Michaelis-Menten behavior with Km,app (HMG-CoA) 28 +/- 3 microM, Km,app (NADPH) 37 +/- 4 microM, and Vm,app 3.9 +/- 0.2 nmol/min mg protein; the transition-state analog lovastatin behaved as a competitive inhibitor with respect to HMG-CoA with Kis 23 nM and a noncompetitive inhibitor toward NADPH with Kii 29 nM. The results are in complete agreement with recent gene cloning and expression studies which showed that T. cruzi HMG-CoA reductase lacks the NH2-terminal membrane-spanning sequence. This is the first demonstration of a soluble eukaryotic HMG-CoA reductase and also the first report on the presence of an enzyme of the isoprenoid biosynthesis pathway in glycosomes.

Animals↗
PubMed1998

Phosphorylation of farnesol in rat liver microsomes: properties of farnesol kinase and farnesyl phosphate kinase.

As farnesol may serve as a nonsterol endogenous regulator of the mevalonate pathway, the possibility that a kinase specific for its phosphorylation is present in the rat liver was investigated. In the 10,000 g supernatant of rat liver, farnesyl monophosphate was synthesized in the presence of ATP. The Km value for farnesol was 2.3 microM. Various detergents inhibited the activity of the enzyme. The farnesol kinase was present in rough and in smooth I microsomes, but not in smooth II microsomes, mitochondria, peroxisomes, Golgi, or plasma membranes. The enzyme was associated with the inner, luminal surface of the vesicles. Further analyses have demonstrated that an enzymatic mechanism exists which catalyzes the phosphorylation of farnesyl-P to farnesyl-PP. Activity of the farnesyl phosphate kinase resulted in the phosphorylation of the monophosphate by CTP but not by ATP, GTP, or UTP. This enzyme is activated by low concentrations of detergents. Treatment with proteases and chemical probes indicate that this second phosphorylation reaction probably takes place on the outer, cytoplasmic surface of microsomal vesicles. These results demonstrate that rat liver microsomes contain two enzymes for the consecutive phosphorylation of farnesol to farnesyl-PP.

Adenosine Triphosphate↗
PubMed1999

Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo.

Bisphosphonates, synthetic compounds which suppress bone resorption, are used in the treatment of skeletal disorders. Their mode of action and intracellular targets have not yet been identified. Recent evidence suggested that enzymes of the mevalonate pathway are the potential targets. In this study, we examined the effect of four potent nitrogen (N)-containing bisphosphonates, clodronate and NH2-olpadronate, an inactive analogue of olpadronate, on isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and protein geranylgeranyl transferase I activity. We found that all N-containing bisphosphonates inhibited isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity dose dependently with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo, whereas clodronate and NH2-olpadronate had no effect. Furthermore, none of the bisphosphonates tested affected geranylgeranyl pyrophosphate synthase or geranylgeranyl transferase I activity. Our study reveals for the first time the intracellular target of N-containing bisphosphonates and supports the view that all bisphosphonates do not share the same molecular mechanism of action.

Animals↗
PubMed1999

Nitrogen-containing bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis.

Nitrogen-containing bisphosphonates are potent bone antiresorptive agents as well as having herbicidal and antiparasitic activity, and are thought to act by inhibiting enzymes of the mevalonate pathway. Using molecular modeling and ab initio quantum chemical calculations, we show that bisphosphonates can act as aza-isoprenoid transition state analogs, thereby inhibiting isoprenoid biosynthesis. The two phosphonate groups of the 1,1-bisphosphonates readily dock into the diphosphate-Mg(2+) binding site in farnesyl diphosphate synthase, while the charged ammonium (or pyridinium or imidazolium) groups act as carbocation transition state analogs, whose binding is stabilized by a cluster of oxygen atoms in the active site cleft, and an overall negative electrostatic potential in this region. Enhanced activity is shown to correlate with increasing van der Waals stabilization due to N-alkylation, or the presence of a charged, planar (sp(2)-hybridized) aromatic residue in the carbocation binding site. These results are of general interest since they suggest a rational approach to bisphosphonate drug design.

Alkyl and Aryl Transferases↗
PubMed2001

Nonorthologous gene displacement of phosphomevalonate kinase.

Phosphomevalonate kinase (PMK; EC 2.7.4.2) catalyzes the phosphorylation of 5-phosphomevalonate into 5-diphosphomevalonate, an essential step in isoprenoid biosynthesis via the mevalonate pathway. So far, two nonorthologous genes encoding PMK have been described, the Saccharomyces cerevisiae ERG8 gene and the human PMK gene. Here, we report that orthologues of ERG8 are present in eubacteria, fungi, and plants, while orthologues of human PMK are found only in animals, indicative of a nonorthologous gene displacement early in animal evolution. This also is reflected by different consensus ATP-binding motifs: a protein kinase motif in the ERG8 orthologues versus a P-loop or Walker A motif in the animal orthologues. The fact that ERG8 orthologues are found in pathogenic eubacteria and fungi but not in man makes them attractive targets for the development of antibacterial and/or antifungal drugs.

Amino Acid Sequence↗
PubMed2005

Plant cells: secondary metabolite heterogeneity and its manipulation.

This chapter proposes the concept of rational manipulation of secondary metabolite heterogeneity in plant cell cultures. The heterogeneity of plant secondary metabolites is a very interesting and important issue because these structure-similar natural products have different biological activities. Both taxoids and ginsenosides are two kinds of preeminent examples in the enormous reservoir of pharmacologically valuable heterogeneous molecules in the plant kingdom. They are derived from the five-carbon precursor isopentenyl diphosphate, produced via the mevalonate or the non-mevalonate pathway. The diterpenoid backbone of taxoids is synthesized by taxadiene synthase and the triterpenoid backbone of ginsenosides is synthesized by dammarenediol synthase or beta-amyrin synthase. After various chemical decorations (oxidation, substitution, acylation, glycosylation, benzoylation, and so on) mediated by P450-dependent monooxygenases, glycosyltransferases, acyltransferases, benzoyltransferases, and other enzymes, the terpenoid backbones are converted into heterogeneous taxoids and ginsenosides with different bioactivities. Although detailed information about accumulation and regulation of individual taxoids or ginsenosides in plant cells is still lacking, remarkable progress has recently been made in the structure and bioactivity identification, biosynthetic pathway, manipulation of their heterogeneity by various methodologies including environmental factors, biotransformation, and metabolic engineering in cell/tissue cultures or in plants. Perspectives on a more rational and efficient process to manipulate production of desired plant secondary metabolites by means of metabolic engineering and "omics"-based approaches (e.g., functional genomics) are also discussed.

Biotechnology↗
PubMed1989

Impact of lemongrass oil, an essential oil, on serum cholesterol.

To test the hypothesis that non-sterol mevalonate pathway end products lower serum cholesterol levels, we asked 22 hypercholesterolemic subjects (315 +/- 9 mg cholesterol/dl) to take a daily capsule containing 140 mg of lemongrass oil, an essential oil rich in geraniol and citral. The paired difference in serum cholesterol levels of subjects completing the 90-day study approached significance (P less than 0.06, 2-tailed t-test). The subjects segregated into two groups, one consisting of 14 subjects resistant to the protocol and the other consisting of 8 subjects who responded. Paired differences in cholesterol level at 30, 60 and 90 d for resistant subjects were +2 +/- 6, +2 +/- 7 and -1 +/- 6 mg/dl; paired differences for the responding subjects were -25 +/- 10 (p less than 0.05), -33 +/- 8 (p less than 0.01) and -38 +/- 10 (p less than 0.025), respectively. The paired difference (+8 +/- 4) in the cholesterol levels of six responders 90 days after the discontinuation of lemongrass oil was not significant.

Cholesterol↗
PubMed2001

Peroxisomes in the apocrine sweat glands of the human axilla and their putative role in pheromone production.

The products of the human apocrine axillary glands contain volatile steroids which act as pheromones. The steroidal structure of these pheromones implies that the axillary glands should be able to synthesize cholesterol which is the essential precursor of these molecules. Since important steps in cholesterol synthesis are localized within peroxisomes, we investigated the occurrence and the putative role of peroxisomes in the axillary glands at protein and mRNA levels by immunocytochemistry, Western blotting, and RT-PCR. Numerous peroxisomes were localized in the cells of the apocrine glands by immunocytochemistry, and the presence of catalase was confirmed by Western blotting and RT-PCR. Additionally, RT-PCR revealed the presence of mRNAs of two peroxisome-associated enzymes of the cholesterol biosynthetic pathway, mevalonate kinase and farnesyl diphosphate synthase. The results suggest that the peroxisomes in the human apocrine axillary glands may play a pivotal role in the biosynthesis of pheromones.

Apocrine Glands↗
PubMed2000

HMG-CoA reductase inhibition: anti-inflammatory effects beyond lipid lowering?

Atherosclerosis has many features of a chronic inflammatory disease. Atherosclerotic lesions contain inflammatory cells like activated T-lymphocytes and macrophages. Systemic markers of inflammation such as white blood cells, C-reactive protein, serum amyloid A, interleukin 6 and soluble adhesion molecules are predictive of future cardiovascular events, even after adjustment for the contribution of established cardiovascular risk factors. Atherogenic lipoprotein particles, in particular modified low-density lipoproteins (LDL), elicit pro-inflammatory responses of cellular elements of the vessel wall, including endothelial dysfunction and activation of monocyte-derived macrophages. Treatment, with HMG-CoA reductase inhibitors has proven the most successful strategy to reduce the concentration of LDL in the circulatory system. These compounds lower LDL cholesterol by inhibiting the mevalonate pathway in the liver, which in turn depletes the regulatory pool of cholesterol and enhances the activity of LDL receptors. Five prospective clinical trials have convincingly demonstrated that HMG-CoA reductase inhibitors can effectively lower the incidence of cardiovascular events in primary and secondary prevention. Post hoc analyses of these trials suggest that the clinical benefit brought about by HMG-CoA reductase inhibitors may not entirely be due to their effect on the levels of circulating lipoproteins. In-vitro observations of anti-inflammatory actions of HMG-CoA reductase inhibitors on vascular cells have been suggested to explain effects beyond lipid-lowering. It is, however, not clear whether these findings are relevant to the in-vivo situation. Further investigation is now necessary in order to determine the relative significance of cholesterol lowering and of ancillary effects to the overall clinical benefit of statin treatment.

Anti-Inflammatory Agents↗
PubMed2006

Isoprenylated proteins.

Isoprenoids are synthesized in all living organisms and are incorporated into diverse classes of end-products that participate in a multitude of cellular processes relating to cell growth, differentiation, cytoskeletal function and vesicle trafficking. In humans, the non-sterol isoprenoids, farnesyl pyrophosphate and geranylgeranyl-pyrophosphate, are synthesized via the mevalonate pathway and are covalently added to members of the small G protein superfamily. Isoprenylated proteins have key roles in membrane attachment and protein functionality, have been shown to have a central role in some cancers and are likely also to be involved in the pathogenesis and progression of atherosclerosis and Alzheimer disease. This review details current knowledge on the biosynthesis of isoprenoids, their incorporation into proteins by the process known as prenylation and the complex regulatory network that controls these proteins. An improved understanding of these processes is likely to lead to the development of novel therapies that will have important implications for human health and disease.

GTPase-Activating Proteins↗
PubMed2002

Effects of risedronate, alendronate, and etidronate on the viability and activity of rat bone marrow stromal cells in vitro.

The effects of risedronate, alendronate, and etidronate were assessed in calcifying fibroblastic colony-forming unit (CFU-f) cultures of rat bone marrow cells in vitro. Biphasic effects on the formation of bone-like colonies were observed. Treatment with high concentrations (10(-5)-10(-4)M) of alendronate and risedronate caused a total inhibition of colony formation whereas etidronate had relatively little effect. It was also found that intermediate concentrations (10(-6)M) of alendronate and risedronate decreased the formation of colonies displaying osteoblastic characteristics such as alkaline phosphatase expression, collagen accumulation, and calcification. At lower concentrations (10(-9)-10(-7)M), risedronate and alendronate increased the formation of fibroblastic colonies, suggesting a mild anabolic effect, however, the formation of colonies with osteoblastic properties was not affected. Treating the cells with a combination of bisphosphonate and 1 mM geranylgeraniol could to some extent abrogate the cytotoxic effects of alendronate or risedronate, suggesting the involvement of the mevalonate pathway. The colony-stimulating activity of these bisphosphonates was, however, unaffected.

Alendronate↗
PubMed2001

Alendronate disturbs vesicular trafficking in osteoclasts.

The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.

Acid Phosphatase↗
PubMed2000

Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase.

The recently discovered non-mevalonate pathway to isoprenoids, which uses glycolytic intermediates, has been modulated by overexpression of Escherichia coli D-1-deoxyxylulose 5-phosphate synthase (DXS) to increase deoxyxylulose 5-phosphate and, consequently, increase the isoprenoid precursor pool in E. coli. Carotenoids are a large class of biologically important compounds synthesized from isoprenoid precursors and of interest for metabolic engineering. However, carotenoids are not ordinarily present in E. coli. Co-overexpression of E. coli dxs with Erwinia uredovora gene clusters encoding carotenoid biosynthetic enzymes led to an increased accumulation of the carotenoids lycopene or zeaxanthin over controls not expressing DXS. Thus, rate-controlling enzymes encoded by the carotenogenic gene clusters are responsive to an increase in isoprenoid precursor pools. Levels of accumulated carotenoids were increased up to 10.8 times the levels of controls not overexpressing DXS. Lycopene accumulated to a level as high as 1333 microg/g dw and zeaxanthin accumulated to a level as high as 592 microg/ g dw, when pigments were extracted from colonies. Zeaxanthin-producing colonies grew about twice as fast as lycopene-producing colonies throughout a time course of 11 days. Metabolic engineering of carbon flow from simple glucose metabolites to representatives of the largest class of natural products was demonstrated in this model system.

Carotenoids↗
PubMed2006

HMG-CoA reductase is regulated by salinity at the level of transcription in Haloferax volcanii.

The moderately halophilic archaeon Haloferax volcanii was surveyed for protein profile changes correlated with growth at high and low salinity. A single polypeptide with an approximate mass of 46 kDa was conspicuously more abundant during growth at high salinity. This protein was identified as HMG-CoA reductase (HMGR), encoded by the hmgR gene. HMGR is a key enzyme in the mevalonate pathway of isoprenoid biosynthesis, the sole route in haloarchaea for lipid and carotenoid production. Enzymatic assays confirmed that HMGR activity is more abundant in cells grown at high salinity. Low salt cultures of H. volcanii contained lower amounts of hmgR transcript compared to cells grown in high salt suggesting that the observed regulation occurs at the level of transcription. Paradoxically, both lipid and carotenoid content decreased in H. volcanii grown at high salinity despite the increased levels of HMGR specific activity. To our knowledge, this is the first report demonstrating that the expression of HMGR is regulated in response to non-optimal salinity in a halophilic archaeon.

Adaptation, Physiological↗
PubMed1992

Effects of mevinolin treatment on tissue dolichol and ubiquinone levels in the rat.

Rats were treated with mevinolin by intraperitoneal injection (15 days) or dietary administration (30 days). The cholesterol, dolichol, dolichyl phosphate and ubiquinone contents of the liver, brain, heart, muscle and blood were then investigated. The cholesterol contents of these organs did not change significantly, with the exception of muscle. Intraperitoneal administration of the drug increases the amount of dolichol in liver, muscle and blood and decreases the dolichyl-P amount in muscle. The same treatment increases the level of ubiquinone in muscle and blood and decreases this value in liver and heart. Oral administration decreases dolichol, dolichyl-P and ubiquinone levels in heart and muscle, while in liver the dolichol level is elevated and ubiquinone level lowered. In brain the amount of dolichyl-P is increased. Intraperitoneal injection of mevinolin also modifies the liver dolichol and dolichyl-P isoprenoid pattern, with an increase in shorter chain polyisoprenes. The levels of dolichol and ubiquinone in the blood do not follow the changes observed in other tissues. Incorporation of [3H]acetate into cholesterol by liver slices prepared from mevinolin-treated rats exhibited an increase, whereas in brain no change was seen. Labeling of dolichol and ubiquinone was increased in both liver and brain, but incorporation into dolichyl phosphate remained relatively stable. The results indicate that mevinolin affects not only HMG-CoA reductase but, to some extent, also affects certain of the peripheral enzymes, resulting in considerable effects on the various mevalonate pathway lipids.

Acetates↗
PubMed1994

Modulations in hepatic branch-point enzymes involved in isoprenoid biosynthesis upon dietary and drug treatments of rats.

Three branch-point enzymes of the mevalonate pathway, farnesyl pyrophosphate synthase, cis-prenyltransferase and squalene synthase were characterized in rat hepatic cytosol, microsomes and peroxisomes isolated from rats after treatment with peroxisome proliferators, inducers of the endoplasmic reticulum or modulators of lipid metabolism. Cholestyramine and phenobarbital induced primarily the cytosolic farnesyl pyrophosphate synthase, whereas clofibrate and phthalates elevated the corresponding peroxisomal activity. cis-Prenyltransferase activities in microsomes were induced 4-5-fold after clofibrate, phthalate and phenobarbital administration, but these same treatments affected the peroxisomal activity to only a limited extent. Squalene synthase activity in microsomes was completely abolished, but the peroxisomal activity was unaffected after administration of cholesterol. On the other hand, clofibrate and phthalate induced only the microsomal activities. Mevinolin treatment greatly increased peroxisomal and cytosolic farnesyl pyrophosphate synthase activities, but not the mitochondrial activity, and the cis-prenyltransferase activities were elevated in peroxisomes, but not in microsomes. These results demonstrate that the branch-point enzymes in cholesterol and dolichol biosynthesis at various cellular locations are regulated differentially and that the capacities of peroxisomes and the endoplasmic reticulum to participate in the synthesis of polyisoprenoid lipids is affected profoundly by treatment with different xenobiotics.

Animals↗
PubMed1994

Lipid peroxidation of microsomal and mitochondrial membranes extracted with n-pentane and reconstituted with ubiquinol, dolichol and cholesterol.

Microsomes and mitochondria prepared from rat liver were extracted with n-pentane, a procedure which does not denature enzyme proteins. Protein and phospholipid were not extracted, but 75-80% of the total dolichol, 80-100% of the ubiquinone and 85-95% of the cholesterol were removed from both organelles by this procedure. Enzymatic and non-enzymatic lipid peroxidation in microsomes and non-enzymatic peroxidation in mitochondria were strongly inhibited when ubiquinol was reinserted into n-pentane-extracted membranes. When reconstitution with dolichol was performed, lipid peroxidation was increased or unchanged, while cholesterol decreased this activity in a concentration-dependent manner. In reconstitution experiments ubiquinol and dolichol together were less inhibitory than ubiquinol alone, whereas cholesterol accentuated the inhibitory effect of ubiquinol. Reconstitution with dolichols of different lengths, dolichyl esters or with alpha-unsaturated polyprenols further demonstrated that dolichol is not an antioxidant. It appears that mevalonate pathway lipids influence lipid peroxidation in membranes by modifying the properties of the bilayer.

Animals↗
PubMed1990

Rates of cholesterol, ubiquinone, dolichol and dolichyl-P biosynthesis in rat brain slices.

Slices from the brain and liver of rats were prepared and upon incubation exhibited a continuous and high capacity for incorporation of radioactive precursors into proteins and lipids. Using [3H]mevalonate as precursor, the rates of biosynthesis of cholesterol, ubiquinone, dolichol and dolichyl-P in brain slices were determined and found to be 5.5, 0.25, 0.0093 and 0.0091 nmol/h/g, respectively. Dolichol and dolichyl-P accumulate to a limited extent, but almost all of these lipids in the brain originate from de novo synthesis. The calculated half-lives for cholesterol, ubiquinone, dolichol and dolichyl-P were 4076, 90, 1006 and 171 h, respectively. The results indicate that lipids formed via the mevalonate pathway in the brain have an active and independently regulated biosynthesis.

Animals↗