Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase

To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.     

Arabidopsis thaliana contains a single gene encoding squalene synthase

Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. Arabidopsis thaliana contains two SQS-annotated genomic sequences, At4g34640 (SQS1) and At4g34650 (SQS2), organized in a tandem array. Here we report that the SQS1 gene is widely expressed in all tissues throughout plant development, whereas SQS2 is primarily expressed in the vascular tissue of leaf and cotyledon petioles, and the hypocotyl of seedlings. Neither the complete A. thaliana SQS2 protein nor the chimeric SQS resulting from the replacement of the 69 C-terminal residues of SQS2 by the 111 C-terminal residues of the Schizosaccharomyces pombe SQS were able to confer ergosterol prototrophy to a Saccharomyces cerevisiae erg9 mutant strain lacking SQS activity. A soluble form of SQS2 expressed in Escherichia coli and purified was unable to synthesize SQ from FPP in the presence of NADPH and either Mg²⁺ or Mn²⁺. These results demonstrated that SQS2 has no SQS activity, so that SQS1 is the only functional SQS in A. thaliana. Mutational studies revealed that the lack of SQS activity of SQS2 cannot be exclusively attributed to the presence of an unusual Ser replacing the highly conserved Phe at position 287. Expression of green fluorescent protein (GFP)-tagged versions of SQS1 in onion epidermal cells demonstrated that SQS1 is targeted to the endoplasmic reticulum (ER) membrane and that this location is exclusively dependent on the presence of the SQS1 C-terminal hydrophobic trans-membrane domain.     

Squalene synthase, a determinant of Raft-associated cholesterol and modulator of cancer cell proliferation

Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.     

Isoprenoids,small GTPases and Alzheimer's disease

The mevalonate pathway is a crucial metabolic pathway for most eukaryotic cells. Cholesterol is a highly recognized product of this pathway but growing interest is being given to the synthesis and functions of isoprenoids. Isoprenoids are a complex class of biologically active lipids including for example, dolichol, ubiquinone, farnesylpyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Early work had shown that the long-chain isoprenoid dolichol is decreased but that dolichyl phosphate and ubiquinone are elevated in brains of Alzheimer′s disease (AD) patients. Until recently, levels of their biological active precursors FPP and GGPP were unknown. These short-chain isoprenoids are critical in the post-translational modification of certain proteins which function as molecular switches in numerous signaling pathways. The major protein families belong to the superfamily of small GTPases, consisting of roughly 150 members. Recent experimental evidence indicated that members of the small GTPases are involved in AD pathogenesis and stimulated interest in the role of FPP and GGPP in protein prenylation and cell function. A straightforward prediction derived from those studies was that FPP and GGPP levels would be elevated in AD brains as compared with normal neurological controls. For the first time, recent evidence shows significantly elevated levels of FPP and GGPP in human AD brain tissue. Cholesterol levels did not differ between AD and control samples. One obvious conclusion is that homeostasis of FPP and GGPP but not of cholesterol is specifically targeted in AD. Since prenylation of small GTPases by FPP or GGPP is indispensable for their proper function we are proposing that these two isoprenoids are up-regulated in AD resulting in an over abundance of certain prenylated proteins which contributes to neuronal dysfunction.     

A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro

Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion.     

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.     

Molecular cloning and expression analysis of a squalene synthase gene from a medicinal plant, Euphorbia pekinensis Rupr.

Euphorbia pekinensis Rupr., which is also known as a medicinal plant, produces a large amount of alkaloids, phytosterols and triterpenes. In this study, we reported on the cDNA cloning and characterization of a novel squalene synthase (SQS) from E. pekinensis. Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol and triterpene biosynthesis. The full length cDNA named EpSQS (Genbank Accession Number JX509735) contained 1,614 bp with an open reading frame of 1,236 bp encoding a polypeptide of 411 amino acids. The deduced amino acid sequence of the EpSQS named EpSQS exhibited a high homology with other plant SQSs, and contained a single domain surrounded by helices. Phylogenetic analysis showed that EpSQS belonged to the plant SQS kingdom. Tissue expression analysis revealed that EpSQS expressed strongly in roots, weakly in stems and leaves, implying that EpSQS was a constitutive expression gene. The recombinant protein was expressed in Escherichia coli and detected by SDS-PAGE and western blot. The high performance liquid chromatography (HPLC) analysis showed that EpSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene.     

Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1

Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway-specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from the hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His(6) tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His(6)-SQS gave a single band at 42 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [(14)C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg(2+) for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the K(m) of FPP is 0.97 +/- 0.10 microM and the k(cat) is 1.74 +/- 0.04 s(-1). Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 +/- 13.6 nM.     

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.     

Is the Reaction Catalyzed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase a Rate-Limiting Step for Isoprenoid Biosynthesis in Plants?

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tabacco (Nicotiana tabaccum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.     

Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.     

Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant development in Arabidopsis

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C(5) units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are <50% of those in wild-type plants, and that the male-sterile phenotype is chemically complemented by squalene, a sterol precursor. In vivo isotope labeling experiments using the atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants.     

Geranylgeranyl Pyrophosphate Is a Potent Regulator of HRD-dependent 3-Hydroxy-3-methylglutaryl-CoA Reductase Degradation in Yeast

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR), the rate-limiting enzymes of sterol synthesis, undergoes feedback-regulated endoplasmic reticulum degradation in both mammals and yeast. The yeast Hmg2p isozyme is subject to ubiquitin-mediated endoplasmic reticulum degradation by the HRD pathway. We had previously shown that alterations in cellular levels of the 15-carbon sterol pathway intermediate farnesyl pyrophosphate (FPP) cause increased Hmg2p ubiquitination and degradation. We now present evidence that the FPP-derived, 20-carbon molecule geranylgeranyl pyrophosphate (GGPP) is a potent endogenous regulator of Hmg2p degradation. This work was launched by the unexpected observation that GGPP addition directly to living yeast cultures caused high potency and specific stimulation of Hmg2p degradation. This effect of GGPP was not recapitulated by FPP, GGOH, or related isoprenoids. GGPP-caused Hmg2p degradation met all the criteria for the previously characterized endogenous signal. The action of added GGPP did not require production of endogenous sterol molecules, indicating that it did not act by causing the build-up of an endogenous pathway signal. Manipulation of endogenous GGPP by several means showed that naturally made GGPP controls Hmg2p stability. Analysis of the action of GGPP indicated that the molecule works upstream of retrotranslocation and can directly alter the structure of Hmg2p. We propose that GGPP is the FPP-derived regulator of Hmg2p ubiquitination. Intriguingly, the sterol-dependent degradation of mammalian HMGR is similarly stimulated by the addition of GGOH to intact cells, implying that a dependence on 20-carbon geranylgeranyl signals may be a common conserved feature of HMGR regulation that may lead to highly specific therapeutic approaches for modulation of HMGR.     

Farnesyl pyrophosphate synthase: a key enzyme in isoprenoid biosynthetic pathway and potential molecular target for drug development

As isoprenoid biosynthetic pathway has gained importance since last few years, key enzymes of this pathway have been characterized and their functional roles in the cell metabolism have been explored using molecular biology approaches. A key enzyme in this pathway is farnesyl pyrophosphate (EC 2.5.1.10) synthase (FPPS) which supplies precursors for the biosynthesis of essential isoprenoids like carotenoids, withanolides, ubiquinones, dolichols, sterols, among others and also helps in farnesylation and geranylation of proteins. It is a chain elongation enzyme which catalyzes head to tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl pyrophosphate (FPP). Recent studies have validated FPPS as a molecular target of bisphosphonates for drug development against tumors as well as human pathogens. The present paper synthesizes the information on characterization, structural and functional relationships, evolution, localization as well as advances on FPPS enzyme as a target for drug development.     

Regulation by heme of sterol uptake in Saccharomyces cerevisiae

The leaky heme mutants G204, G216, and G214 are shown to accumulate exogenous sterols. Unlike hem mutants which have complete blocks in the heme pathway, these strains do not require ergosterol, methionine, or unsaturated fatty acids for growth. The addition of aminolevulinic acid to the growth medium inhibited sterol uptake in G204 96% but had only a slight effect on sterol uptake by strains G214 and G216. Sterol uptake in all three strains was inhibited 83-94% when cells were grown in the presence of hematin. Sterol analysis of these strains grown in the presence and absence of either aminolevulinic acid or hematin revealed that saturation of the cell membrane with ergosterol was not responsible for the dramatic decrease in sterol uptake. These results suggest that sterol uptake by yeast cells is controlled by heme, and explain the non-viability of yeast strains that are heme competent and auxotrophic for sterols.