Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase

The cDNA for human squalene monooxygenase, a key enzyme in the committed pathway for cholesterol biosynthesis, was amplified from a human liver cDNA library and cloned, and the protein was expressed in Escherichia coli and purified. Kinetic analysis of the purified enzyme revealed an apparent K(m) for squalene of 7.7 microM and an apparent k(cat) of 1.1 min(-1). For FAD the apparent K(m) is 0.3 microM, consistent with a loosely bound flavin. The apparent K(m) for NADPH-cytochrome P450 reductase, the requisite electron transfer partner, is 14 nM. The amount of reductase needed for maximal activity is about threefold less than the amount of squalene monooxygenase present in the assay; thus, electron transfer to the monooxygenase is not likely to be rate limiting. Previous reports have implicated inhibition of this enzyme as the cause of a peripheral demyelination seen in weanling rats fed a diet containing tellurium. As no data were available for humans, the ability of a number of tellurium and related elemental compounds to inhibit the recombinant human enzyme was examined. Tellurite, tellurium dioxide, selenite, and selenium dioxide were inhibitory; the tellurium compounds were more potent than the selenium compounds, as indicated by their IC(50) values (17 and 37 microM, respectively). Kinetic analysis of the inhibition by tellurite suggests multiple sites of interaction with the enzyme in a noncompetitive manner with respect to squalene.     

Tellurite Specifically Affects Squalene Epoxidase: Investigations Examining the Mechanism of Tellurium-Induced Neuropathy

Abstract: A peripheral neuropathy characterized by a transient demyelinating/remyelinating sequence results when young rats are fed a tellurium-containing diet. The neuropathy occurs secondary to a systemic block in cholesterol synthesis. Squalene accumulation suggested the lesion was at the level of squalene epoxidase, a microsomal monooxygenase that uses NADPH cytochrome P450 reductase to receive its necessary reducing equivalents from NADPH. We have now demonstrated directly specificity for squalene epoxidase; our in vitro studies show that squalene epoxidase is inhibited 50% in the presence of 5 µ M tellurite, the presumptive in vivo active metabolite. Under these conditions, the activities of other monooxygenases, aniline hydroxylase and benzo( a )pyrene hydroxylase, were inhibited less than 5%. We also present data suggesting that tellurite inhibits squalene epoxidation by interacting with highly susceptible -SH groups present on this monooxygenase. In vivo studies of specificity were based on the compensatory response to feeding of tellurium. Following tellurium intoxication, there was up-regulation of squalene epoxidase activity both in liver (11-fold) and sciatic nerve (fivefold). This induction was a specific response, as demonstrated in liver by the lack of up-regulation following exposure to the nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also measured the microsomal monooxygenase activities of aniline hydroxylase and benzo( a )pyrene hydroxylase. Although they were induced following phenobarbital exposure, activities of these monooxygenases were not affected following tellurium intoxication, providing further evidence of specificity of tellurium intoxication for squalene epoxidase.     

Inhibition of human squalene monooxygenase by selenium compounds

Selenosis in animals is characterized by a variety of neurological abnormalities, but the chemical species of selenium and the molecular targets that mediate this neurotoxicity are unknown. We have previously shown that selenite is a potent inhibitor of squalene monooxygenase, the second enzyme in the committed pathway for cholesterol biosynthesis; inhibition of this enzyme by dimethyltellurium leads to a peripheral demyelinating neuropathy similar to that seen in selenosis. To evaluate the role methylation plays in selenium toxicity, we examined the ability of three methylselenium compounds, methylselenol, dimethylselenide, and trimethylselenonium iodide, to inhibit purified recombinant human squalene monooxygenase. IC(50) values for methylselenol (95 microM) and dimethylselenide (680 microM) were greater than that previously obtained for selenite (37 microM), and inhibition by trimethylselenonium iodide was evident only at concentrations above 3 mM. Inhibition by methylselenol as well as by selenite was slow and irreversible, suggestive of covalent binding to the enzyme, and thiol-containing compounds could prevent and reverse this inhibition, indicating that these compounds were reacting with sulfhydryl groups on the protein. Monothiols such as glutathione and beta-mercaptoethanol provided better protection than did dithiols, suggesting that these selenium compounds bind to only one of the two proposed vicinal cysteines on squalene monooxygenase. Unexpectedly, the inhibition by selenite was significantly enhanced by dithiols, indicating that a more toxic species, possibly selenide, was formed in the presence of these dithiol reductants.     

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.     

Squalene monooxygenase - a target for hypercholesterolemic therapy

Squalene monooxygenase catalyzes the epoxidation of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compounds of these organisms and squalene epoxidation is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively, which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clinical use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clinical practice. After many years of use of statins there is some clinical evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, especially in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the future.     

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.     

Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase in vitro and, unexpectedly, cholesterol synthesis in cell culture. Because squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, we investigated the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway. Substitution of [(14)C]mevalonate for [(14)C]acetate in McARH7777 hepatoma cells expressing SPF reduced the 1.8-fold increase in cholesterol synthesis by half, suggesting that SPF acted on or prior to mevalonate synthesis. This conclusion was supported by the finding that substitution with [(14)C]mevalonate completely blocked an SPF-induced increase in squalene synthesis. Evaluation of 2,3-oxidosqualene synthesis from [(14)C]mevalonate demonstrated that SPF also stimulated squalene monooxygenase (1.3-fold) in hepatoma cells. Immunoblot analysis showed that SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels, indicating a direct effect on enzyme activity. Addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold, and the SPF-concentration/activation curve paralleled that for the SPF-mediated stimulation of squalene monooxygenase. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.     

Tellurium-Induced Alterations in 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene Expression and Enzyme Activity: Differential Effects in Sciatic Nerve and Liver Suggest Tissue-Specific Regulation of Cholesterol Synthesis

The demyelination of peripheral nerves that results from exposure of developing rats to tellurium is due to inhibition of squalene epoxidase, a step in cholesterol biosynthesis. In sciatic nerve, cholesterol synthesis is greatly depressed, whereas in liver, some compensatory mechanism maintains normal levels of cholesterol synthesis. This tissue specificity was further explored by examining, in various tissues, gene expression and enzyme activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Exposure to tellurium resulted in pronounced increases in both message levels and enzyme activity in liver, the expected result consequent to up-regulation of this enzyme in response to decreasing levels of intracellular sterols. In contrast to liver, levels of mRNA and enzyme activity in sciatic nerve were both decreased during the tellurium-induced demyelinating period. The temporal pattern of changes in 3-hydroxy-3-methylglutaryl-CoA reductase message levels in sciatic nerve seen following exposure to tellurium was similar to the down-regulation seen for mRNA specific for PNS myelin proteins. Possible mechanisms for differential control of cholesterol biosynthesis in sciatic nerve and liver are discussed.     

Contrasting effects of selenite and tellurite on lipoamide dehydrogenase activity suggest a different biological behaviour of the two chalcogens

The effects of selenite and tellurite on the mammalian enzyme lipoamide dehydrogenase were compared. Selenite acts as a substrate of lipoamide dehydrogenase in a process requiring the presence of lipoamide. In contrast, tellurite is a potent inhibitor, effective in the low micromolar range. The inhibitory effect of tellurite on lipoamide dehydrogenase is partially reverted by dithiothreitol indicating the participation of the thiol groups of the enzyme. Tellurite, but not selenite, stimulates the diaphorase activity of lipoamide dehydrogenase. In a mitochondrial matrix protein preparation, which contains lipoamide dehydrogenase, an inhibitory action similar to that observed on the purified enzyme was also elicited by tellurite. Human embryonic kidney cells (HEK 293 T) treated with tellurite show a partial inhibition of lipoamide dehydrogenase. In addition to the toxicological implications of tellurium compounds, the reported results suggest that tellurite and its derivatives can be used as potential tools for studying biochemical reactions.     

Binding of tellurium to hepatocellular selenoproteins during incubation with inorganic tellurite: consequences for the activity of selenium-dependent glutathione peroxidase

The metallic group XVIa elements selenium and tellurium possess remarkably similar chemical properties. However, unlike selenium, tellurium is not an essential micronutrient and, indeed, induces both acute and chronic toxicity in a variety of species. Despite this, very little is known of the molecular mechanisms of toxicity of tellurium, particularly with respect to potential chemical interactions with selenium-containing components in the cell. In this work we describe a novel interaction of inorganic tellurite with hepatocellular selenoproteins, particularly with selenium-dependent glutathione peroxidase. The accumulation of (121Te)-tellurite into cultured primary rat liver hepatocytes was shown to be much more rapid than that of (75Se)-selenite on a molar basis. Neither the uptake of (121Te)-tellurite nor of (75Se)-selenite was affected by a large molar excess of the unlabelled counterpart, respectively. Interestingly, separation of the hepatocellular proteins on continuous pH denaturing gels demonstrated clear binding of radiolabelled tellurium to a number of protein bands, including one at 23 and one at 58 kDa, which corresponded to proteins readily labelled in cells treated with (75Se)-selenite. The binding of (121Te) to these proteins was insensitive to reduction with mercaptoethanol and not affected by pre-treatment of the cells with cycloheximide. When purified selenium-dependent glutathione peroxidase was treated directly with (121Te)-tellurite, the protein became labelled in an analogous manner to that achieved in intact cells. This was not affected by coincubation of the enzyme with (121Te)-tellurite and one or both of its substrates. Additionally, incubation of the peroxidase with tellurite effectively inhibited its ability to catalyse glutathione-dependent reduction of hydrogen peroxide. These data suggest that inorganic tellurite delivers tellurium to the intracellular milieu in a form capable of binding to some intracellular selenoproteins and at least in the case of glutathione peroxidase, cause inhibition of catalytic activity. The nature of the binding seems not to be due to the insertion of tellurocysteine into the protein and the insensitivity to reductive cleavage with mercaptoethanol seems to preclude the formation of stable telluro-selenides in the proteins. These data may offer alternative explanations for the established toxicity of tellurium via disruption of selenoprotein function, particularly by the induction of intracellular oxidative stress by the inhibition of Se-dependent glutathione peroxidase.     

Green tea polyphenols: novel and potent inhibitors of squalene epoxidase

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50) = 0.69 microM), (-)-gallocatechin-3-O-gallate (GCG) (IC(50) = 0.67 microM), (-)-epicatechin-3-O-gallate (ECG) (IC(50) = 1.3 microM), and theasinensin A (IC(50) = 0.13 microM), were found to be potent and selective inhibitors of rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. On the other hand, flavan-3-ols without galloyl group at C-3 did not show significant enzyme inhibition. It was demonstrated for the first time that the cholesterol lowering effect of green tea may be attributed to their potent SE inhibition activities. Inhibition kinetics revealed that EGCG inhibited SE in noncompetitive (K(I) = 0.74 microM), and non-time-dependent manner. The potent enzyme inhibition would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction.     

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.     

Lipid Droplets in Schwann Cells During Tellurium Neuropathy Are Derived from Newly Synthesized Lipid

Exposure of weanling rats to a diet containing elemental tellurium results in a peripheral neuropathy characterized by segmental demyelination and minimal axonal degeneration. One of the earliest ultrastructural abnormalities in tellurium neuropathy is an increased number of cytoplasmic lipid droplets in myelinating Schwann cells. The pathogenesis of these lipid droplets was investigated using light and electron microscopic autoradiography. Nerve lipids were either "prelabeled" with [3H]acetate via in vivo intraneural injection 3 days before a 2-day exposure to tellurium, or "postlabeled" via in vivo intraneural injection or in vitro incubation with [3H]acetate following a 2-day exposure to tellurium. In the prelabeled nerves, myelin became heavily labeled, but the tellurium-induced cytoplasmic lipid droplets were rarely labeled. In the postlabeled nerves, the tellurium-induced cytoplasmic lipid droplets were the most heavily labeled structures within the nerve. These data indicate that the tellurium-induced lipid droplets in Schwann cells are derived from newly synthesized lipid rather than from the early breakdown and internalization of myelin lipids. The earliest biochemical abnormality observed in tellurium neuropathy is an inhibition of cholesterol synthesis at the squalene epoxidase step. This leads to an accumulation of squalene within the nerve. We conclude that the cytoplasmic lipid droplets in Schwann cells contain this accumulated lipid.     

Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase

Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital yet potentially toxic lipid. Squalene monooxygenase (SM) catalyzes the first oxygenation step in cholesterol synthesis, acting on squalene before cyclization into the basic steroid structure. Using model cell systems, we found that cholesterol caused the accumulation of the substrate squalene, suggesting that SM may serve as a flux-controlling enzyme beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, considered as rate limiting). Cholesterol accelerated the proteasomal degradation of SM which required the N-terminal domain, partially conserved in vertebrates but not in lower organisms. Unlike HMGR, SM degradation is not mediated by Insig, 24,25-dihydrolanosterol, or side-chain oxysterols, but rather by cholesterol itself. Importantly, SM's N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins. Furthermore, proteasomal inhibition almost totally eliminated squalene accumulation, highlighting the importance of this degradation mechanism for the control of SM and suggesting this as a possible control point in cholesterol synthesis.     

Pyridine-2,6-bis(thiocarboxylic acid) produced by Pseudomonas stutzeri KC reduces and precipitates selenium and tellurium oxyanions

The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H(2)S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.     

Hepatic cytochrome P450 reductase-null mice reveal a second microsomal reductase for squalene monooxygenase

Squalene monooxygenase is a microsomal enzyme that catalyzes the conversion of squalene to 2,3(s)-oxidosqualene, the immediate precursor to lanosterol in the cholesterol biosynthesis pathway. Unlike other flavoprotein monooxygenases that obtain electrons directly from NAD(P)H, squalene monooxygenase requires a redox partner, and for many years it has been assumed that NADPH-cytochrome P450 reductase is this requisite redox partner. However, our studies with hepatic cytochrome P450-reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase. Inhibition studies with antibody to P450 reductase indicate that this second reductase supports up to 40% of the monooxygenase activity that is obtained with microsomes from normal mice. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol; this lanosterol metabolite also accumulates in the livers of CPR-null mice, indicating that cholesterol synthesis is blocked at lanosterol demethylase, a cytochrome P450.     

Phosphorylation of supernatant protein factor enhances its ability to stimulate microsomal squalene monooxygenase

Supernatant protein factor is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway. The mechanism of stimulation is poorly understood, although supernatant protein factor belongs to a family of lipid-binding proteins that includes Sec14p and alpha-tocopherol transfer protein. Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a relatively weak ability to activate microsomal squalene monooxygenase, we investigated the possibility that cofactors or post-translational modifications were necessary for full activity. Addition of ATP to rat liver cytosol increased supernatant protein factor activity by more than 2-fold and could be prevented by the addition of inhibitors of protein kinases A and C. Incubation of purified recombinant supernatant protein factor with ATP and protein kinases A or C delta similarly increased activity by more than 2-fold. Addition of protein phosphatase 1 gamma, a serine/threonine phosphatase, to rat liver cytosol reduced activity by 50%, suggesting that supernatant protein factor is partially phosphorylated in vivo. To determine whether dietary cholesterol influenced the phosphorylation state, cytosols were prepared from livers of rats fed a high fat diet. Although supernatant protein factor activity was reduced by more than one-half, it could not be restored by the addition of ATP or protein kinase C delta with ATP, suggesting that dietary cholesterol reduced the expression of this protein. Supernatant protein factor thus appears to be regulated both post-translationally through phosphorylation and at the level of expression. Phosphorylation may provide a means for the rapid short term modulation of cholesterol synthesis.     

Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters

n-Alkyl esters (ethyl, octyl, dodecyl, and cetyl) of gallic acid were evaluated as enzyme inhibitors of recombinant rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. Dodecyl (6) (IC(50) = 0.061 microM) showed the most potent inhibition, which was far more potent than those of previously reported naturally occurring gallocatechins. Octyl gallate (5) (IC(50) = 0.83 microM) and cetyl gallate (7) (IC(50) = 0.59 microM) also showed good inhibition, while gallic acid (IC(50) = 73 microM) itself was not so active. In addition, chemically synthesized galloyl ester of cholesterol (9) (IC(50) = 3.9 microM), farnesol derivative (10) (IC(50) = 0.57 microM), and dodecyl galloyl amide (8) (IC(50) = 3.0 microM) were also potent inhibitors of SE. Inhibition kinetics revealed that dodecyl gallate inhibited SE in competitive (K(I) = 0.033 microM) and no-time-dependent manner. The potent inhibition of the flavin monooxygenase would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the epoxidation reaction.     

Pyridine-2,6-Bis(Thiocarboxylic Acid) Produced by Pseudomonas stutzeri KC Reduces and Precipitates Selenium and Tellurium Oxyanions

The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H₂S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.     

Squalene monooxygenase: a journey to the heart of cholesterol synthesis

Squalene monooxygenase (SM) is a vital sterol synthesis enzyme across eukaryotic life. In yeast, it is a therapeutic target for treating certain fungal infections, and in mammals it is a rate-limiting enzyme that represents a key control point in the cholesterol synthesis pathway. SM introduces an oxygen atom to squalene, which becomes the signature oxygen of the hydroxyl group in cholesterol. Our knowledge of SM has advanced tremendously since its initial cloning and characterization. Early research developed mammalian SM inhibitors to target SM for cholesterol-lowering purposes. The substrate squalene has gained considerable interest for its health benefits and in nanomedicine for delivery of drugs. More recently, SM has been implicated as a key dysregulated component in certain cancers. In this review, we summarize our present knowledge of SM, focusing on the regulation of SM and the gene encoding it, SQLE. Furthermore, we offer insights into the role of SM across different organisms and its significance in human health and disease.