Identification of the Arabidopsis dry2/sqe1‐5 mutant reveals a central role for sterols in drought tolerance and regulation of reactive oxygen species

Squalene epoxidase enzymes catalyse the conversion of squalene into 2,3‐oxidosqualene, the precursor of cyclic triterpenoids. Here we report that the Arabidopsis drought hypersensitive/squalene epoxidase 1‐5 (dry2/sqe1‐5) mutant, identified by its extreme hypersensitivity to drought stress, has altered stomatal responses and root defects because of a point mutation in the SQUALENE EPOXIDASE 1 (SQE1) gene. GC‐MS analysis indicated that the dry2/sqe1‐5 mutant has altered sterol composition in roots but wild‐type sterol composition in shoots, indicating an essential role for SQE1 in root sterol biosynthesis. Importantly, the stomatal and root defects of the dry2/sqe1‐5 mutant are associated with altered production of reactive oxygen species. As RHD2 NADPH oxidase is de‐localized in dry2/sqe1‐5 root hairs, we propose that sterols play an essential role in the localization of NADPH oxidases required for regulation of reactive oxygen species, stomatal responses and drought tolerance.     

环氧角鲨烯合成通路在大肠杆菌中的构建和优化

三萜类化合物是一类广泛应用于医药、保健和化妆品等行业的天然产物,具有巨大的商业价值.生物合成三萜类化合物依赖于环氧角鲨烯的高效合成.角鲨烯环氧化酶是整个合成途径中的关键酶,其催化NADPH依赖的环氧化反应将角鲨烯转变为环氧角鲨烯.通过筛选不同来源的角鲨烯环氧化酶,截短的大鼠角鲨烯环氧化酶(RnSETC)在大肠杆菌Esc...     

Squalene epoxidase as hypocholesterolemic drug target revisited

Therapeutic success of statins has distinctly established inhibition of de novo hepatic cholesterol synthesis as an effective approach to lower plasma LDL-cholesterol, the major risk factor for atherosclerosis and coronary heart disease. Statins inhibit HMG CoA reductase, a rate limiting enzyme which catalyses conversion of HMG CoA to mevalonic acid. However, in this process statins also inhibit the synthesis of several non-sterols e.g. dolichols and ubiquinone, which are implicated in side effects observed with statins. This prompted many major pharmaceutical companies in 1990s to target selective cholesterol synthesis beyond farnesyl pyrophosphate. The enzymes squalene synthetase, squalene epoxidase and oxidosqualene cyclase were identified as potential targets. Though inhibitors of these enzymes have been developed, till date no compound has been reported to have entered clinical trials. We evaluated the literature to understand merits and demerits of pursuing squalene epoxidase as a target for hypocholesterolemic drug development. Squalene epoxidase catalyses the conversion of squalene to 2,3-oxidosqualene. Although it has been extensively exploited for antifungal drug development, it has received little attention as a target for hypocholesterolemic drug design. This enzyme though recognized in the early 1970s was cloned 25 years later. This enzyme is an attractive step for pharmacotherapeutic intervention as it is the secondary rate limiting enzyme and blocking cholesterol synthesis at this step may result in accumulation of only squalene which is known to be stable and non toxic. Synthesis of several potent, orally bioavailable inhibitors of squalene epoxidase has been reported from Yamonuchi, Pierre Fabre and Banyu pharmaceuticals. Preclinical studies with these inhibitors have clearly demonstrated the potential of squalene epoxidase inhibitors as hypocholesterolemic agents. Hypochloesterolemic therapy is intended for prolonged duration and safety is an important determinant in clinical success. Lack of clinical trials, despite demonstrated preclinical efficacy by oral route, prompted us to evaluate safety concerns with squalene epoxidase inhibitors. In dogs, NB-598, a potent competitive squalene epoxidase inhibitor has been reported to exhibit signs of dermatitis like toxicity which has been attributed by some reviewers to accumulation of squalene in skin cells. Tellurium, a non-competitive inhibitor of squalene epoxidase has been associated with neuropathy in weanling rats. On the other hand, increased plasma levels of squalene in animals and humans (such as occurring subsequent to dietary olive oil or squalene administration) are safe and associated with beneficial effect such as chemoprevention and hypocholesterolemic activity. In our view, high circulating levels of squalene epoxidase inhibitor may be responsible for dermatitis and neuropathy. Competitive inhibition and pharmacokinetic profile minimizing circulating plasma levels (e.g. by hepatic sequestration and high first pass metabolism) could be important determinants in circumventing safety concerns of squalene epoxidase inhibitors. Recently, cholesterol-lowering effect of green tea has been attributed to potent squalene epoxidase inhibition, which can be consumed in much higher doses without toxicological effect. These facts strengthen optimism for developing clinically safe squalene epoxidase inhibitors. Put in perspective squalene epoxidase appears to be undervalued target which merits attention for development of better hypocholesterolemic drugs.     

Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.     

Role of supernatant protein factor and anionic phospholipid in squalene uptake and conversion by microsomes

Supernatant protein factor (SPF), a cytosolic protein (Mr = 47,000) stimulates microsomal squalene epoxidase activity 4- to 10-fold in the presence of anionic phospholipid such as phosphatidylglycerol (PG) (Saat, Y., and Bloch, K. (1976) J. Biol. Chem. 251, 5155-5160). This effect has been ascribed to substrate translocation from inactive to active pools within the membrane of the endoplasmic reticulum (Friedlander, E. J., Caras, I. W., Lin, L. F. H., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). Here we show that SPF and PG also stimulate squalene uptake per se by microsomes as well as stimulate squalene epoxidase. Microsomes preloaded with substrate in the presence of SPF and PG show full epoxidase activity. They do not require further addition of these factors during enzyme assay. Addition of SPF and PG to assay mixtures containing microsomes preloaded with substrate in the presence of SPF and PG did not further increase epoxidase activity. We also show that PG tightly binds to microsomes. This binding of PG is essential for the response of microsomal epoxidase to SPF. Solubilized microsomal enzymes have been reconstituted and show high epoxidase activity. In this system, SPF and PG do not stimulate the conversion of squalene into products.     

质膜NADPH氧化酶的活化与H2O2的产生介导内源激发子诱导人参悬浮细胞中PAL活性的提高与人参皂甙的积累

利用纤维素酶降解人参(Panax ginseng C．A．Meyer)悬浮细胞的细胞壁制备了内源激发子(CDW)。CDW体外诱导了游离人参细胞质膜NADPH氧化酶的活性,激发了活体人参悬浮细胞产生H2O2。CDW还可以诱导提高苯丙氨酸解氨酶(PAL)活性,促进人参鲨烯环氧酶基因(sqe)的转录与人参皂甙的积累。NADPH氧化酶的抑制剂不仅可以抑制CDW体外诱导的质膜NADPH活性而且还可以抑制CDW诱导人参细胞产生H2O2。进而,这些抑制剂还可以抑制CDW诱导PAL活性的提高,以及sqe的转录与人参皂甙的合成。过氧化氢酶与H2O2的粹灭剂也可以抑制CDW激发产生的这些诱导效应。上述结果表明CDW激发质膜NADPH氧化酶的活化与H2O2的产生在介导CDW诱导人参细胞抗性反应中,包括PAL活性的提高与人参皂甙的积累,起了重要的信号转导作用。     

A squalene epoxidase from Nigella sativa participates in saponin biosynthesis and mediates terbinafine resistance in yeast

Squalene epoxidase catalyzes the formation of 2,3-oxidosqualene from squalene and in plants is the last enzyme common to all biosynthetic pathways leading to an array of triterpene derivatives like phytosterols, brassinosteroid phytohormones or saponins. In this work, we present a squalene epoxidase gene (NSSQE1) from the triterpene saponin producing plant Nigella sativa. The gene product showed a high degree of homology to functional squalene epoxidases (SQEs) from Arabidopsis thaliana and was able to complement SQE deficient yeast that harboured a knockout mutation in the underlying erg1 gene. Moreover, the expression of the NSSQE1 gene in ERG1 wild type yeast revealed that NSSQE1 conferred resistance towards terbinafine, an inhibitor of fungal SQEs. The latter suggested that a terbinafine-dependent NSSQE1 selection marker system can be developed for yeast. The gene NSSQE1 was ubiquitously expressed in all plant tissues analysed, including roots where no triterpene saponins are produced. Therefore, we argue that NSSQE1 is a housekeeping gene for triterpene metabolism in Nigella sativa. Similar to triterpene saponins, NSSQE1 was up-regulated by methyl jasmonate in leaves and should also be functionally involved in saponin biosynthesis in Nigella sativa.     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Fungal elicitor induces singlet oxygen generation, ethylene release and saponin synthesis in cultured cells of Panax ginseng C. A. Meyer

Singlet oxygen is a high-energy molecular oxygen species. As one of the most active intermediates involved in chemical and biochemical reactions, singlet oxygen plays essential roles in plant responses to UV and strong light. Here, we report that Cle, an elicitor derived from fungal cell walls, induces the generation of singlet oxygen in cell cultures of ginseng, Panax ginseng. Cle treatment also triggers the activation of plasma membrane NADPH oxidase and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), subsequently leading to ethylene release and increased saponin synthesis, as shown by increased mRNA expression of squalene synthase (SQS) and squalene epoxidase (SQE), and accumulation of beta-amyrin synthase (beta-AS). Suppression of Cle-induced singlet oxygen generation or inhibition of ethylene production blocks saponin synthesis, whereas treatment of ginseng cells with ethylene or singlet oxygen induces the synthesis of saponin. Together, these results indicate that Cle-induced production of both singlet oxygen and ethylene is required for saponin synthesis, and that singlet oxygen may function upstream of ethylene during Cle-induced saponin synthesis.     

Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system.

Microsomal squalene epoxidase has previously been solubilized with Triton X-100 and resolved into fractions, FA and FB, by DEAE-cellulose chromatography (Ono T. and Bloch K (1975) J biol. Chem. 250, 1571-1579). It has now been found that FB is identical with NADPH-cytochrome c reductase (denoted FPT, EC 1.6.2.3). Although both NADPH and NADH served as electron donors, the former was preferred for squalene epoxidase activity in the reconstituted system of FA and FB. FB is characterized by its ability to reduce cytochrome c by NADPH. In place of FB, partially purified FPT was tested for its ability to support squalene epoxidation in the presence of FA. A stepwise purification of the deoxycholate-solubilized FPT yielded an increase in specific FPT activity with a parallel increase in squalene epoxidase activity. Bromelain-solubilized FPT was less effective. Rabbit antisera preparations to the purified FPT solubilized with trypsin were shown to inhibit concomitantly FPT activity and squalene epoxidase activity. These observations support the concept that squalene epoxidation is primarily mediated via a flavoprotein, NADPH-cytochrome c reductase, and a terminal oxidase, squalene epoxidase, which is distinct from cytochrome P-450.     

Role of Organotellurium Species in Tellurium Neuropathy

Exposure of weanling rats to a diet containing 1% elemental tellurium causes segmental demyelination of peripheral nerve, and an inhibition of squalene epoxidase. This inhibition is thought to be the mechanism of action leading to demyelination. Tellurite appears to be the active inhibitory species in a cell-free system but the active species in vivo is unknown. We examined potassium tellurite (K₂TeO₃) and three organotellurium compounds for their ability to inhibit squalene epoxidase in Schwann cell cultures and to induce demyelination in weanling rats. K₂TeO₃ had no effect on squalene epoxidase activity in cultured Schwann cells and caused no demyelination in vivo. All three organotellurium compounds caused inhibition of squalene epoxidase in vitro and caused demyelination in vivo. (CH₃)₂TeCl₂ was the most potent of these compounds and its neuropathy most resembled that caused by elemental tellurium. These data are consistent with the hypothesis that tellurium-induced demyelination is a result of squalene epoxidase inhibition and suggest that a dimethyltelluronium compound may be the neurotoxic species presented to Schwann cells in vivo.     

Inactivation and activation of various membranal enzymes of the cholesterol biosynthetic pathway by digitonin

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

解脂耶氏酵母中羽扇豆醇合成途径的构建与调控

羽扇豆醇因其具有抗癌抗炎等生理活性而广泛应用于医药领域.本研究分别利用源自木榄和蓖麻的羽扇豆醇合酶(LUS)在解脂耶氏酵母(Yarrowia lipolytica)中构建生物合成羽扇豆醇途径(GLU-1、GLU-2),并由对该途径中关键限速酶3-羟基-3-甲基戊二酰辅酶a还原酶(tHMGR)和异戊烯基二磷酸异构酶(ID...     

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

Inhibition of squalene epoxidase by allylamine antimycotic compounds. A comparative study of the fungal and mammalian enzymes.

The inhibition of squalene epoxidase by the allylamine antimycotic agents naftifine and compound SF 86-327 was investigated, with particulate enzyme preparations from the pathogenic yeasts Candida albicans and Candida parapsilosis and from rat liver. Both naftifine and compound SF 86-327 were potent inhibitors of the Candida epoxidases and showed apparently non-competitive kinetics with respect to the substrate squalene. The Ki values for naftifine and compound SF 86-327 in the C. albicans system were 1.1 microM and 0.03 microM respectively. The C. parapsilosis enzyme was slightly more sensitive to inhibition. Varying the concentrations of cofactors or the soluble cytoplasmic fraction (S200) had no effect on the inhibition. The epoxidase from rat liver was much less sensitive (Ki for compound SF 86-327 was 77 microM). The inhibition was also qualitatively different from that in Candida, being competitive with respect to squalene and also with respect to the S200 fraction. S200 fraction derived from C. albicans also antagonized the inhibition of the epoxidase from liver, but the liver S200 fraction did not affect inhibition of the Candida enzyme by compound SF 86-327. There was no evidence for an irreversible or mechanism-based inhibition of either the fungal or the mammalian epoxidase. The selective inhibition of squalene epoxidase was sufficient to account for the known antimycotic action of the compounds.     

Tellurium Blocks Cholesterol Synthesis by Inhibiting Squalene Metabolism: Preferential Vulnerability to This Metabolic Block Leads to Peripheral Nervous System Demyelination

Inclusion of 1.1% elemental tellurium in the diet of postweanling rats produces a peripheral neuropathy due to a highly synchronous primary demyelination of sciatic nerve; this demyelination is followed closely by remyelination. Sciatic nerves from animals fed tellurium for various times were removed and incubated ex vivo for 1 h with [14C]acetate, and radioactivity incorporated into individual lipid classes was determined. In nerves from rats exposed to tellurium, there was a profound and selective block in the conversion of radioactive acetate to cholesterol. Another radioactive precursor, [3H]water, gave similar results. We suggest that tellurium feeding inhibits squalene epoxidase activity and that the consequent lack of cholesterol destabilizes myelin, thereby causing destruction of the larger internodes. Ex vivo incubation experiments were also carried out with liver slices. As with nerve, tellurium feeding caused accumulation in squalene of label from radioactive acetate, whereas labeling of cholesterol was greatly inhibited. Unexpectedly, however, incorporation of label from [3H]water into both squalene and cholesterol was increased. Relevant is the demonstration that liver was the primary site of bulk accumulation of squalene, which accounted for 10% of liver dry weight at 5 days. Thus, accumulation of squalene (and other mechanisms, possibly including up-regulation of cholesterol biosynthetic pathways) drives squalene epoxidase activity at normal levels in liver even in the presence of inhibitors of this enzyme. This is reflected by continuing incorporation of [3H]water into cholesterol; incorporation of this precursor takes place at many of the postsqualene biosynthetic steps for sterol formation. [14C]Acetate entering the sterol pathway before squalene in liver is greatly diluted in specific activity when it reaches the large squalene pool, and thus increased squalene epoxidase activity does not transfer significant 14C label to sterols. In contrast to the situation with liver, synthesis of sterols is markedly depressed in sciatic nerve, and squalene does not accumulate to high levels.