BIOLOGY OF SQUALENE MONOOXYGENASE

The conversion of squalene, a 30-carbon linear isoprenoid, to lanosterol, a tetracyclic compound, occurs in two steps that were first elucidated in the laboratories of Corey, van Tamelen, and Bloch in the late 1960's.¹  Yamamoto and Bloch¹ showed that the first step, catalyzed by squalene monooxygenase, required both the microsomal and cytosolic fraction of liver, along with NADPH and O₂.  Konrad Bloch had earlier received the Nobel Prize in Physiology or Medicine in 1964 along with Feodor Lynen "for their discoveries concerning the mechanism and regulation of cholesterol and fatty acid metabolism". 

Later studies from Bloch's laboratory² showed that squalene monooxygenase is bound to the endoplasmic reticulum of cells in association with NADPH-cytochrome P450 reductase, its electron transfer partner.  The enzyme has a loosely-bound FAD (flavin) group.  The cytosolic fraction was shown to be composed of phospholipid and a 45 kDa protein termed 'supernatant protein factor' or 'sterol carrier protein₁'.³  Supernatant protein factor (SPF) was recently cloned and shown to be a member of the cytosolic lipid binding/transfer protein family, including yeast phosphatidylinositol transfer protein (Sec14p).⁴  The exact role of SPF remains unclear; it can be replaced by the nonionic detergent Triton X100 for in vitro assays.  

Activity studies indicate that squalene epoxidase is expressed at very low or negligible levels in most non-cholesterolgenic tissues, and is found in greatest abundance in the liver, followed by the gut, skin, and neural tissue.⁵  Its low abundance and low specific activity suggest that squalene monooxygenase may be the rate-limiting component in cholesterol biosynthesis. 

Squalene monooxygenase is regulated at the transcriptional level in response to sterol levels in the cell.  Although HMG-CoA reductase is traditionally considered to be the regulated step in cholesterol synthesis, it is now clear that squalene synthase and squalene monooxygenase are also important regulatory points.  As shown in the figure to the right, squalene monooxygenase (SE) expression can be increased by lowering blood cholesterol levels with cholestryramine, a dietary lipid binder, or blocking cholesterol synthesis at HMG-CoA reductase with lovastatin.⁶  Squalene monooxygenase, like HMG-CoA reductase, exhibits diurnal variation in activity, with activity highest during the night.⁶ 

Recent studies from Teruo Ono's laboratory have shown that enzyme activity is regulated by changes in gene transcription in response to sterol levels, including the oxysterol 25-hydroxycholesterol.⁷  25-Hydroxycholesterol, which also down-regulates HMG-CoA reductase and LDL receptor expression, may be a physiological feedback regulator of the cholesterol biosynthesis pathway. 

The human squalene monooxygenase gene (SQLE) is located on chromosome 8q24.13.⁸ 

Current inhibitors of cholesterol biosynthesis in man block HMG-CoA reductase, the second enzyme in this multi-step pathway.  Because this has been shown to decrease the synthesis of isoprenoid compounds that are involved in cell physiology, growth, and regulation, there has been continued interest in developing inhibitors that act more specifically on cholesterol synthesis.  The most effective inhibitor of mammalian squalene monooxygenases to date is NB-598, developed at Banyu Pharmaceutical Co.  This fungal-derived natural compound is a competitive inhibitor of squalene monooxygenase in human HepG2 cells with a Ki of 0.68 nM.⁹  It effectively reduces serum cholesterol in dogs with no apparent adverse effects.^9,10  No studies have yet been reported in man. 

A variety of chemical compounds found in edible and medicinal plants have recently been shown to be potent and selective inhibitors of squalene monooxygenase.  Ikuro Abe's laboratory has shown that green tea polyphenols are particularly potent of inhibitors of the recombinant rat enzyme.¹¹  The presence of a galloyl group (3,4,5-trihydroxybenzoyl) was necessary for inhibition; epigallocatechin-3-O-gallate (EGCG), the major green tea polyphenol, had a Ki of 0.74 µM.  The major metabolites of EGCG were also inhibitory.  Although a typical cup of green tea contains 100 mg of EGCG, the low bioavailability and 2-3 hr half-life suggests that significant tea consumption would be necessary to obtain therapeutic levels.¹²  EGCG is a noncompetitive inhibitor of squalene monooxygenase, and may act by scavenging the reactive oxygen species formed at the active site of the enzyme (the flavin 4a hydroperoxide).  Other plant extracts that contain galloyl esters, including rhubarb and the Chinese herb fo-ti (Polygonum multiflorum), have also been found to inhibit squalene monooxygenase and reduce serum cholesterol.¹¹  Additional studies by Abe's group have identified several synthetic galloyl esters as potent inhibitors of squalene monooxygenase, including dodecyl gallate, with a Ki of 33 nM.¹³  Dodecyl gallate, and other synthetic alkylgalloyl esters, are widely used as antioxidant food additives.  

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenol found in grape skins and red wine, has also been reported to lower cholesterol and prevent cardiovascular disease;¹⁴ we have found that resveratrol is a modest  inhibitor of squalene monooxygenase, with a Ki of 35 µM with respect to squalene.¹⁵  As with the galloyl esters, resveratrol is a reversible, noncompetitive inhibitor of the enzyme. 

Garlic is also reputed to lower blood cholesterol and have a variety of beneficial cardiovascular effects; we find that a 0.5% (final concentration) aqueous extract of fresh garlic inhibits greater than 90% of recombinant human squalene monooxygenase activity in vitro.¹⁶  Because inhibition by garlic is irreversible, it is likely to act by a different mechanism than the polyphenols; one or more of the many oxidized sulfur compounds in garlic is likely to bind to and permanently inactivate the enzyme.  S-allylcysteine is abundant in garlic and is one of the more potent inhibitors; it is a principal component of some commercial garlic preparations (e.g, Kyolic).  

Several inhibitors of squalene monooxygenase in yeast are currently on the market.  Terbinafine (Lamisil®) and Naftifine (Naftin®) show good specificity for the fungal enzyme without inhibiting human squalene monooxygenase.¹⁷  Both are fungicidal, interfering with cell membrane synthesis and preventing growth.  Terbinafine is effective both topically and orally.

1. Cory EJ, Russey WE, Ortiz de Montellano PR.  2,3-oxidosqualene, an intermediate in the biological synthesis of sterols from squalene.  J Am Chem Soc 1966 Oct 20; 88(20):4750-1.  Van Tamelen EE, Willett JD, Clayton RB, Lord KE.  Enzymic conversion of squalene 2,3-oxide to lanosterol and cholesterol.  J Am Chem Soc 1966 Oct 20; 88(20):4752-4.  Yamamoto S, Bloch K.  Studies on squalene epoxidase of rat liver.  J Biol Chem 1970 Apr 10; 245(7):1670-4
2. Ono T, Bloch K.  Solubilization and partial characterization of rat liver squalene epoxidase. J Biol Chem 1975 Feb 25; 250(4):1571-9
3. Tai HH, Bloch K.  Squalene epoxidase of rat liver.  J Biol Chem 1972 Jun 25; 247(12):3767-73.  Ferguson JB, Bloch K.  Purification and properties of a soluble protein activator of rat liver squalene epoxidase.  J Biol Chem 1977 Aug 10;252(15):5381-5.  Srikantaiah MV, Hansbury E, Loughran ED, Scallen TJ.  Purification and properties of sterol carrier protein₁. J Biol Chem 1976 Sep 25; 251(18):5496-504.  
4. Shibata N, Arita M, Misaki Y, Dohmae N, Takio K, Ono T, Inoue K, Arai H.  Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis.  Proc Natl Acad Sci U S A 2001 Feb 27;98(5):2244-2249 [abstract]
5. Astruc M, Tabacik C, Descomps B, de Paulet AC.  Squalene epoxidase and oxidosqualene lanosterol-cyclase activities in cholesterogenic and non-cholesterogenic tissues.  Biochim Biophys Acta 1977 Apr 26; 487(1):204-11
6. Satoh T, Hidaka Y, Kamei T.  Regulation of squalene epoxidase activity in rat liver.  J Lipid Res 1990 Nov; 31(11):2095-101 [abstract]
7. Nakamura Y, Sakakibara J, Izumi T, Shibata A, Ono T.  Transcriptional regulation of squalene epoxidase by sterols and inhibitors in HeLa cells.  J Biol Chem 1996 Apr 5; 271(14):8053-6 [abstract]
8. Nagai M, Sakakibara J, Wakui K, Fukushima Y, Igarashi S, Tsuji S, Arakawa M, Ono T.  Localization of the squalene epoxidase gene (SQLE) to human chromosome region 8q24.1.  Genomics 1997 Aug 15; 44(1):141-3 [abstract]
9. Horie M, Tsuchiya Y, Hayashi M, Iida Y, Iwasawa Y, Nagata Y, Sawasaki Y, Fukuzumi H, Kitani K, Kamei T.  NB-598: a potent competitive inhibitor of squalene epoxidase.  J Biol Chem 1990 Oct 25; 265(30):18075-8 [abstract]
10. Horie M, Sawasaki Y, Fukuzumi H, Watanabe K, Iizuka Y, Tsuchiya Y, Kamei T.  Hypolipidemic effects of NB-598 in dogs.  Atherosclerosis 1991 Jun; 88(2-3):183-92 [abstract]
11. Abe I, Seki T, Umehara K, Miyase T, Noguchi H, Sakakibara J, Ono T.  Green tea polyphenols: novel and potent inhibitors of squalene epoxidase.  Biochem Biophys Res Commun 2000 Feb 24;268(3):767-71 [abstract].  Abe I, Seki T, Noguchi H, Kashiwada Y.  Galloyl esters from rhubarb are potent inhibitors of squalene epoxidase, a key enzyme in cholesterol biosynthesis.  Planta Med 2000 Dec;66(8):753-6 [abstract]
12. Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, Balentine DA, Yang CS.  Analysis of plasma and urinary tea polyphenols in human subjects.  Cancer Epidemiol Biomarkers Prev 1995 Jun;4(4):393-9 [abstract]; Chen L, Lee MJ, Li H, Yang CS.  Absorption, distribution, elimination of tea polyphenols in rats.  Drug Metab Dispos 1997 Sep;25(9):1045-50 [abstract]; Zhu M, Chen Y, Li RC.  Oral absorption and bioavailability of tea catechins.  Planta Med 2000 Jun;66(5):444-7 [abstract]
13. Abe I, Seki T, Noguchi H.  Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters.  Biochem Biophys Res Commun 2000 Apr 2;270(1):137-40  [abstract]
14. Fremont L.  Biological effects of resveratrol.  Life Sci 2000 Jan 14;66(8):663-73 [abstract].  German JB, Walzem RL.  The health benefits of wine.  Annu. Rev. Nutr. 2000, Vol. 20: 561-93 [abstract]
15. Laden BP, Porter, TD.  Resveratrol inhibits human squalene monooxygenase.  Nutrition Res. 2001 May; 21(5):747-753
16. Gupta N, Porter TD.  Garlic and garlic-derived compounds inhibit human squalene monooxygenase.  J Nutr 2001 Jun;131(6):1662-7 [abstract]
17. Ryder NS.  Terbinafine: mode of action and properties of the squalene epoxidase inhibition.  Br J Dermatol 1992 Feb; 126 Suppl 39:2-7 [abstract]

More information on squalene monooxygenase:

• The squalene monooxygenase family and related enzymes

  • Squalene monooxygenase sequences

  • Alignment of squalene monooxygenases

  • Other flavoproteins related to squalene monooxygenase

  • Comparison of squalene monooxygenase to p-hydroxybenzoate hydroxylase

  • The 3-dimensional structure of p-hydroxybenzoate hydroxylase
     

• Enzymology of squalene monooxygenase

  • Mechanism

  • Interaction with cytochrome P450 reductase

  • Interaction with squalene and FAD
     

• Inhibition by tellurium and selenium compounds

  • Chemistry and Toxicity of Tellurium and Selenium

  • Inhibition of Squalene Monooxygenase by Tellurium and Selenium

  • Tellurium and Selenium Compounds React with Cysteines on Squalene Monooxygenase
     

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