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PubMed2004

Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae.

Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.

Amino Acid Sequence↗
PubMed1993

The deficiency of sterol biosynthesis in Saccharomyces cerevisiae affects the synthesis of glycosyl derivatives of dolichyl phosphates.

Mutants deficient in sterol (thermosensitive ergosterol auxotrophs) erg 8, 9, 12 and heme synthesis hem 1, 12 were screened for the level of free dolichol and dolichyl phosphate synthesized in the mevalonate pathway as well as for the activity of dolichyl phosphate-dependent glycosyl transferases. The amount of DolP synthesized via CTP-dependent phosphorylation was the same in mutants and parental strains. However, mannosylation and glucosylation of endogenous dolichyl phosphates in ergosterol mutants was about four times lower compared to parental strains, while the same reactions carried out with exogenous Dol24P reached 80% of the level observed in parental strains indicating that activities of DolPMan and DolPGlc synthases are not the rate-limiting factors. It is postulated that the de novo synthesis of DolP is impaired in the ergosterol mutants. Moreover, a block in the ergosterol branch of the metabolic pathway (erg 9) causes an increase in the de novo synthesis of dolichyl phosphate.

Dolichol Monophosphate Mannose↗
PubMed1999

Farnesol and geranylgeraniol prevent activation of caspases by aminobisphosphonates: biochemical evidence for two distinct pharmacological classes of bisphosphonate drugs.

Recently, advances have been made in understanding the molecular mechanisms by which bisphosphonate drugs inhibit bone resorption. Studies with the macrophage-like cell line J774 have suggested that alendronate, an amino-containing bisphosphonate, causes apoptosis by preventing post-translational modification of GTP-binding proteins with isoprenoid lipids. However, clodronate, a nonaminobisphosphonate, does not inhibit protein isoprenylation but can be metabolized intracellularly to a cytotoxic, beta-gamma-methylene (AppCp-type) analog of ATP. These observations raise the possibility that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R2 side chain. We addressed this question by directly comparing the ability of three aminobisphosphonates (alendronate, ibandronate, and pamidronate) and three nonaminobisphosphonates (clodronate, etidronate, and tiludronate) to inhibit protein isoprenylation and activate caspase-3-like proteases or to be metabolized to AppCp-type nucleotides by J774 cells. All three aminobisphosphonates inhibited protein isoprenylation and activated caspase-3-like proteases. Apoptosis and caspase activation after 24-h treatment with the aminobisphosphonates could be prevented by addition of farnesol or geranylgeraniol, confirming that these bisphosphonates inhibit the metabolic mevalonate pathway. No AppCp-type metabolites of the aminobisphosphonates could be detected by mass spectrometry. The three nonaminobisphosphonates did not inhibit protein isoprenylation or cause activation of caspase-3-like proteases, but were incorporated into AppCp-type nucleotides. Taken together, these observations clearly demonstrate that bisphosphonate drugs can be divided into two pharmacological classes: the aminobisphosphonates, which act by inhibiting protein isoprenylation, and the less potent nonaminobisphosphonates, which act through the intracellular accumulation of AppCp-type metabolites.

Adenosine Triphosphate↗
PubMed2005

Antileishmanial activity of the terpene nerolidol.

The activity of nerolidol, a sesquiterpene used as a food-flavoring agent and currently under testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs, was evaluated against Leishmania species. Nerolidol inhibited the growth of Leishmania amazonensis, L. braziliensis, and L. chagasi promastigotes and L. amazonensis amastigotes with in vitro 50% inhibitory concentrations of 85, 74, 75, and 67 microM, respectively. The treatment of L. amazonensis-infected macrophages with 100 microM nerolidol resulted in 95% reduction in infection rates. Inhibition of isoprenoid biosynthesis, as shown by reduced incorporation of [2-(14)C]mevalonic acid (MVA) or [1-(14)C]acetic acid precursors into dolichol, ergosterol, and ubiquinone, was observed in nerolidol-treated promastigotes. This drug effect can be attributed to the blockage of an early step in the mevalonate pathway, since incorporation of the precursor [1(n)-(3)H]farnesyl pyrophosphate in polyisoprenoids is not inhibited by nerolidol. L. amazonensis-infected BALB/c mice were treated with intraperitoneal doses of 100 mg/kg/day for 12 days or topically with 5 or 10% ointments for 4 weeks. Significant reduction of lesion sizes in nerolidol treated mice was observed for both treatment routes. However, long-term follow up indicated that the disease was not cured in this highly susceptible animal model. Nonetheless, the in vitro activity of nerolidol against these parasites may prove a useful tool for the development of new drugs for the treatment of leishmaniasis. In addition, biosynthesis of dolichols with 11 and 12 isoprene units was identified in Leishmania, as described for other trypanosomatids and Apicomplexa.

Animals↗
PubMed1997

Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-R) is the major rate-limiting enzyme of the mevalonate pathway in many organisms, including yeasts. In the yeast Saccharomyces cerevisiae, there are two isoenzymes of HMG-R (Hmg1p and Hmg2p). Both consist of an anchoring transmembrane domain and a catalytic domain. We have removed the known controlling features of HMG-R by overproducing the catalytic domain of Hmg1p. This overproduction leads to an enhancement of squalene production, implying that HMG-R has been deregulated. The enhancement is apparent under semianaerobic and aerobic conditions. Despite the increase in squalene production, the amount of ergosterol produced by the HMG-R-overproducing yeast was not increased. This result suggests the presence of another regulatory step between squalene and ergosterol formation. Squalene levels generated by cells overproducing the catalytic domain of HMG-R were estimated to be up to 10 times those produced by wild-type cells. The enhancement in squalene production coincided with a reduction in growth rate. This reduction may be a direct consequence of the buildup of high concentrations of squalene and presqualene intermediates of the pathway.

Aerobiosis↗
PubMed1998

Sterol uptake in Saccharomyces cerevisiae heme auxotrophic mutants is affected by ergosterol and oleate but not by palmitoleate or by sterol esterification.

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17alpha-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.

Animals↗
PubMed1999

Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase.

Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3'-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44 degrees C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the V(max) of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in K(m)s. The E. coli protein requires Mg(2+) or Mn(2+) for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.

Carbon-Carbon Double Bond Isomerases↗
PubMed1997

Protein prenylation is required for aldosterone-stimulated Na+ transport.

Aldosterone stimulation of transcellular Na+ flux in polarized epithelial cells is dependent on at least one transmethylation reaction, but the substrate of this signaling step is unknown. Because it is clear that the majority of cellular protein methylation occurs in conjunction with protein prenylation, we examined the importance of prenylation to aldosterone-stimulated Na+ transport in the A6 cell line. Lovastatin, an inhibitor of the first committed step of the mevalonate pathway, inhibits the natriferic effect of aldosterone but does not inhibit insulin-stimulated Na+ flux. The addition of a farnesyl group does not appear to be involved in aldosterone's action. Neither alpha-hydroxyfarne-sylphosphonic acid, an inhibitor of farnesyl:protein transferase, nor N-acetyl-S-farnesyl-L-cysteine, an inhibitor of farnesylated protein methylation, inhibits the hormone-induced increase in Na+ transport. In contrast, N-acetyl-S-geranyl-geranyl-L-cysteine, an inhibitor of geranylgeranyl protein methylation, completely abolishes the aldosterone-induced increase in Na+ flux with no effect on insulin-mediated Na+ transport or cellular protein content. These data indicate that methylation of a geranylgeranylated protein is involved in aldosterone's natriferic action.

Acetylcysteine↗
PubMed2006

Molecular mechanisms of action of bisphosphonates: current status.

PURPOSE: Bisphosphonates are currently the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia. These compounds have high affinity for calcium ions and therefore target bone mineral, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. EXPERIMENTAL DESIGN: This article reviews the pharmacology of bisphosphonates and the relationship between chemical structure and antiresorptive potency. We also describe new insights into their intracellular molecular mechanisms of action, methods for assessing the effects of bisphosphonates on protein prenylation, and their potential as direct antitumor agents. RESULTS: Nitrogen-containing bisphosphonates act intracellularly by inhibiting farnesyl diphosphate synthase, an enzyme of the mevalonate pathway, thereby preventing prenylation of small GTPase signaling proteins required for normal cellular function. Inhibition of farnesyl diphosphate synthase also seems to account for their antitumor effects observed in vitro and for the activation of gamma,delta T cells, a feature of the acute-phase response to bisphosphonate treatment in humans. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that seems to involve primarily the formation of cytotoxic metabolites in osteoclasts. CONCLUSIONS: Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts in vivo but could also have direct effects on other cell types, such as tumor cells. After >30 years of clinical use, their molecular mechanisms of action on osteoclasts are finally becoming clear but their exact antitumor properties remain to be clarified.

Animals↗
PubMed2001

Onset, time course, and persistence of increased haemodialysis-induced breath isoprene emission.

Recent findings of increased isoprene emission in the exhaled breath of patients undergoing haemodialysis and experimental evidence of the potential toxic and cancerogenic effects of isoprene hydrocarbon led us to assess how long haemodialysis patients are exposed to how much isoprene after a single haemodialysis session. Patients with end-stage renal failure on regular 4-hour (from 08.00 to 12.00 h) maintenance haemodialysis three times weekly were monitored. The breath isoprene content was analyzed by gas chromatography. Intrapatient evaluations were performed by collecting samples before, during, and immediately after the haemodialysis session, during the following hours, and on the following nondialysis day. The breath isoprene content increased in all patients. Isoprene overproduction showing a biphasic pattern was first detected soon after the dialysis session ended. These data show that haemodialyzed patients seem to be consistently exposed to high endogenous isoprene concentrations. The mechanisms and implications of this endogenous isoprene overproduction need to be elucidated with regard to the mevalonic pathway and in the physiopathological setting of the uraemia-dialysis syndrome.

Adult↗
PubMed2003

Tissue-specific utilization of menaquinone-4 results in the prevention of arterial calcification in warfarin-treated rats.

The effects of vitamin K (phylloquinone: K1 and menaquinone-4: MK-4) on vascular calcification and their utilization in the arterial vessel wall were compared in the warfarin-treated rat model for arterial calcification. Warfarin-treated rats were fed diets containing K1, MK-4, or both. Both K1 and MK-4 are cofactors for the endoplasmic reticulum enzyme gamma-glutamyl carboxylase but have a structurally different aliphatic side chain. Despite their similar in vitro cofactor activity we show that MK-4 and not K1 inhibits warfarin-induced arterial calcification. The total hepatic K1 accumulation was threefold higher than that of MK-4, whereas aortic MK-4 was three times that of K1. The utilization of K1 and MK-4 in various tissues was estimated by calculating the ratios between accumulated quinone and epoxide species. K1 and MK-4 were both equally utilized in the liver, but the aorta showed a more efficient utilization of MK-4. Therefore, the observed differences between K1 and MK-4 with respect to inhibition of arterial calcification may be explained by both differences in their tissue bioavailability and cofactor utilization in the reductase/carboxylase reaction. An alternative explanation may come from an as yet hypothetical function of the geranylgeranyl side chain of MK-4, which is a structural analogue of geranylgeranyl pyrophosphate and could interfere with a critical step in the mevalonate pathway.

Animals↗
PubMed2004

Role of isoprenoids in cytoskeleton integrity and albumin endocytosis by opossum kidney cells.

BACKGROUND: The actin cytoskeleton has been increasingly implicated in endocytic events that are involved in the reabsorption of filtered protein by the proximal tubule. Isoprenylated small G proteins have emerged as key regulators of the actin cytoskeleton. This study examines the role of isoprenoid intermediates in organization of the cytoskeleton, and the effect of modification of the cytoskeleton on albumin endocytosis. METHODS: The effect of lovastatin on cytoskeleton morphology of opossum kidney cells (OK cells) was determined by staining with fluorescent isothiocyanate (FITC)-phalloidin followed by examination using confocal microscopy. Quantitative effects on albumin binding and uptake were determined using a well-established method. RESULTS: Inhibition of isoprenoid synthesis by lovastatin led to morphological disruption of the cytoskeleton with a concentration-dependent decrease in albumin uptake into OK cells. Addition of mevalonate, but not cholesterol, ameliorated the effects of lovastatin on the cytoskeleton and albumin uptake. Selective inhibition of isoprenoid intermediates with a farnesyltransferase inhibitor suggests that geranylgeranylated proteins mediate cytoskeleton organization. CONCLUSION: These results confirm the importance of cytoskeleton integrity in albumin endocytosis in renal proximal tubules. While the present data suggest that synthesis of isoprenoids via the mevalonate pathway is a critical step in maintenance of the cytoskeleton, the role of individual small G proteins in control of the cytoskeleton and other endocytic events is yet to be defined.

Albumins↗
PubMed2003

Circadian rhythms in Neurospora crassa: farnesol or geraniol allow expression of rhythmicity in the otherwise arrhythmic strains frq10, wc-1, and wc-2.

In Neurospora crassa, the circadian rhythm can be seen in the bd (band) strain as a series of "bands" or conidiation (spore-forming) regions on the surface of an agar medium. Certain mutations at 3 different genes (frq, wc-1, or wc-2) lead to the loss of the circadian rhythm. In this study, it was found that the addition of 10(-4) to 10(-5) M of geraniol or farnesol restored rhythmic banding to strains that lack a circadian rhythm due to mutations in any 1 of these 3 genes. These 3 conditionally arrhythmic strains now exhibited robust, free-running conidiation rhythms. Their rhythms were neither temperature-compensated nor obviously sensitive to light, so the full properties of a circadian rhythm were not restored. At 20 degrees C, in growth tubes, farnesol treatment gave periods of 28, 26, and 22 h for the frq10, wc-1, and wc-2 strains, respectively. Geraniol treatment at 20 degrees C gave periods of 23, 25.5, and 24.5 h for the frq10, wc-1, and wc-2 strains, respectively. A PRC for temperature pulses (1 h, 20 to 40 degrees C) for the frq10 strain grown in the presence of geraniol showed strong resetting (type 0), suggesting that a temperature-sensitive oscillator was present. Farnesol and geraniol are related to known intermediates in the steroid (or mevalonate) pathway. These data are interpreted in terms of a 2-oscillator model, in which farnesol/geraniol activate or amplify a remaining oscillator (a postulated frq-less oscillator).

Activity Cycles↗
PubMed2006

The effect of bisphosphonates on gene expression: GAPDH as a housekeeping or a new target gene?

BACKGROUND: RT-PCR has been widely used for the analysis of gene expression in many systems, including tumor samples. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) has been frequently considered as a constitutive housekeeping gene and used to normalize changes in specific gene expression. However, GAPDH has been shown to be up-regulated in many cancers and down-regulated by chemotherapic drugs. Bisphosphonates, potent inhibitors of bone resorption, have recently shown a direct and indirect antitumor effect in vitro and in animal models. They exert their effects mainly by inhibiting the mevalonate pathway but also by modulating the expression of many genes not only in osteoclasts but also in cancer cells. METHODS: We evaluated GAPDH gene expression by real time RT PCR in breast (MCF-7 and T47D) and prostate (PC3 and DU-145) cancer cell lines treated with amino and non-amino bisphosphonates. RESULTS: Our results showed that amino-bisphosphonates significantly decrease in a dose-dependent manner the expression of GAPDH gene. CONCLUSION: Therefore, GAPDH is inaccurate to normalize mRNA levels in studies investigating the effect of bisphosphonates on gene expression and it should be avoided. On the other hand, this gene could be considered a potential target to observe the effects of bisphosphonates on cancer cells.

Breast Neoplasms↗
PubMed2002

Statins as modulators of bone formation.

The use of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) to reduce serum cholesterol is well described. However, the recent finding that statins have direct effects on bone was unexpected. A number of epidemiological studies have recently been published that explore the effects of statins on bone mineral density and risk of fracture in humans. Statins may act by directly stimulating the expression of bone morphogenetic protein-2 and increasing osteoblast differentiation or, like nitrogen-containing bisphosphonates, may have effects on the mevalonate pathway that leads to inhibition of osteoclast activity and osteoblast apoptosis. In addition, the demonstration that statins can inhibit inflammation and encourage angiogenesis offers other possibilities for action.

Bone Density↗
PubMed2001

Development of bisphosphonates.

Bisphosphonates are synthetic compounds characterized by a P[bond]C[bond]P group, and are thus analogs of inorganic pyrophosphate. They are used in medicine mainly to inhibit bone resorption in diseases like osteoporosis, Paget's disease and tumor bone disease. They have been used for over a century in industry, and only in 1968 was it shown that bisphosphonates have biological effects. These effects consist mainly of an inhibition of bone resorption and, when given in large amounts, an inhibition of ectopic and normal calcification. While the latter effect is the consequence of a physical-chemical inhibition of calcium phosphate crystal formation, the former is due to a cellular effect involving both apoptosis of the osteoclasts and a destruction of the osteoclastic cytoskeleton, inducing a decrease in osteoclast activity. The biochemical basis of these effects for the nitrogen-containing compounds is an inhibition of the mevalonate pathway caused by the inhibition of farnesylpyrophosphate synthase, which leads to a decrease of the formation of isoprenoid lipids such as farnesylpyrophosphate and geranylgeranylpyrophosphate. The other bisphosphonates are incorporated into the phosphate chain of ATP-containing compounds so that they become non-hydrolyzable. The new P[bond]C[bond]P-containing ATP analogs inhibit cell function and may lead to apoptosis and death of osteoclasts.

Antineoplastic Agents↗
PubMed2003

The use of zoledronic acid in the management of metastatic bone disease and hypercalcaemia.

Zoledronic acid is a potent, third generation, nitrogen-containing bisphosphonate, licensed for the management of skeletal metastases and hypercalcaemia of malignancy, both of which cause considerable morbidity. In the preclinical setting, zoledronic acid has demonstrated superior potency regarding inhibition of osteolysis and reduction of hypercalcaemia as compared with other bisphosphonates. Clinical trials have indicated that zoledronic acid is superior to pamidronate in suppressing osteolysis and in reducing hypercalcaemia of malignancy. Its main mechanism of action is induction of osteoclast apoptosis through inhibition of the mevalonate pathway. Zoledronic acid has also demonstrated direct anti-tumour activity both in vitro and in animal models, suggesting it may be of benefit in preventing the formation of bone metastases. Clinical trials are in progress, assessing the benefit of zoledronic acid in the adjuvant setting in both breast and prostate cancer.

Bone Neoplasms↗
PubMed2006

Increased cholesterol biosynthesis and hypercholesterolemia in mice overexpressing squalene synthase in the liver.

Squalene synthase (SS) is the first committed enzyme for cholesterol biosynthesis, located at a branch point in the mevalonate pathway. To examine the role of SS in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirus-mediated gene transfer. Overexpression of SS increased de novo cholesterol biosynthesis with increased 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase activity, in spite of the downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrations of LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily caused by increased hepatic production of cholesterol-rich VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339. mRNA expression of LDLR was decreased, suggesting that defective LDL clearance contributed to the development of hypercholesterolemia. Curiously, the liver was enlarged, with a larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism through modulation of HMG-CoA reductase activity and cholesterol biosynthesis.

Acyl Coenzyme A↗