7-Dehydrocholesterol–dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome
Barbara U. FitzkyFabian F. MoebiusHitoshi AsaokaHeather Waage-BaudetLi‐Wen XuGuorong XuNobuyo MaedaKimberly D. KluckmanSylvia HillerHongwei YuAshok K. BattaSarah SheferTai C. ChenGerald SalenKathleen K. SulikRobert SimoniGene C. NessHartmut GlossmannShailendra B. PatelG.S. Tint
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Smith–Lemli–Opitz syndrome
Proteolysis
The Smith-Lemli-Opitz syndrome is a disorder of morphogenesis resulting from an enzymatic defect in the last step of cholesterol metabolism (reduction of 7-dehydrocholesterol). Analysis of the defective gene and identification of mutations therein have paved the way for the study of the molecular genetics of the disorder which is caused by numerous different mutations. Future efforts should identify a postulated intracellular signalling activity sterol intermediates, isolate proteins that govern the sterol traffic between intracellular compartments, structurally characterize the enzyme δ7-sterol reductase defective in the Smith-Lemli-Opitz syndrome and investigate the pathomechanism of sterol depletion-induced dysmorphogenesis.
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Sterol O-acyltransferase
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Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Δ7-sterol reductase (EC 1.3.1.21 ), suspected to be defective in the Smith–Lemli–Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Δ7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C 7–8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC 50 0.013 μM), BM15766 (IC 50 1.2 μM), and triparanol (IC 50 14 μM). Our work paves the way to clarify whether a defect in the Δ7-sterol reductase gene underlies the Smith–Lemli–Opitz syndrome.
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Smith–Lemli–Opitz syndrome
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Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7–/– mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.
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Mevalonate pathway
Coenzyme A
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Smith Lemli Opitz syndrome (SLOS) caused by a deficit of 3beta-hydroxysterol-Delta7 reductase was the first sterol deficit described with multiple malformations. The lack of specificity of many morphological abnormalities detected by ultrasound and their frequency have justified routine screening of amniotic fluid (AF) for sterols by GC-MS. The examination contributes to an improved knowledge of the sterol status in the fluid.A series of sterol profiles is collated here. Accumulation of 7- and 8-dehydrocholesterol are diagnostic for SLOS. However, a number of other sterols have also been detected by GC-MS in control AF and their presence may be confusing.In addition to cholesterol, the level of which varies as function of the gestational age, lathosterol is present together with trace amounts of 7- and 8-dehydrocholesterol and other precursors such as desmosterol, lanosterol, and dimethylsterol. Phytosterols are also present in 70% of AF samples that have been tested. Besides SLOS, GC-MS examination of amniotic fluid can detect various sterol deficits associated with malformations (lathosterolosis, desmosterolosis, X-linked chondrodysplasia, and particular Antley-Bixler syndrome). Practical conclusions support GC-MS as a routine method to investigate skeletal and central nervous system malformations.
Smith–Lemli–Opitz syndrome
Desmosterol
Lathosterol
Amniocentesis
Cholestanol
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Smith–Lemli–Opitz syndrome
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Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7–/– mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.
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Coenzyme A
Mevalonate pathway
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Minor aberrant pathways of cholesterol biosynthesis normally produce only trace levels of abnormal sterol metabolites but may assume major importance when an essential biosynthetic step is blocked. Cholesta-5,8-dien-3β-ol, its Δ5,7 isomer, and other noncholesterol sterols accumulate in subjects with the Smith-Lemli-Opitz syndrome (SLOS), a severe developmental disorder caused by a defective Δ7 sterol reductase gene. We have explored the formation and metabolism of unsaturated sterols relevant to SLOS by incubating tritium-labeled Δ5,8, Δ6,8, Δ6,8(14), Δ5,8(14), and Δ8 sterols with rat liver preparations. More than 60 different incubations were carried out with washed microsomes or the 10,000 g supernatant under aerobic or anaerobic conditions; some experiments included addition of cofactors, fenpropimorph (a Δ8–Δ7 isomerase inhibitor), and/or AY-9944 (a Δ7 reductase inhibitor). The tritium-labeled metabolites from each incubation were identified by silver ion high performance liquid chromatography on the basis of their coelution with unlabeled authentic standards, as free sterols and/or acetate derivatives. The Δ5,8 sterol was converted slowly to cholesterol via the Δ5,7 sterol, which also slowly isomerized back to the Δ5,8 sterol. The Δ6,8 sterol was metabolized rapidly to cholesterol by an oxygen-requiring pathway via the Δ7,9(11), Δ8, Δ7, and Δ5,7 sterols as well as by an oxygen-independent route involving initial isomerization to the Δ5,7 sterol. The Δ8 sterol was partially metabolized to Δ5,8, Δ6,8, Δ7,9(11), and Δ5,7,9(11) sterols when isomerization to Δ7 was blocked. The combined results were used to formulate a scheme of normal and aberrant biosynthetic pathways that illuminate the origin and metabolic fate of abnormal sterols observed in SLOS and chondrodysplasia punctata.—Ruan, B., J. Tsai, W. K. Wilson, and G. J. Schroepfer, Jr. Aberrant pathways in the late stages of cholesterol biosynthesis in the rat: origin and metabolic fate of unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome. J. Lipid Res. 2000. 41: 1772–1782.
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Cholestanol
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Hydroxymethylglutaryl-CoA reductase
Coenzyme A
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Abstract There is epidemiologic evidence that the hydrophilic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor pravastatin increases the incidence of some extrahepatic cancers, although this finding has been attributed to chance. We hypothesize that pravastatin is able to promote the development of cancer by causing an induction of HMG-CoA reductase and, hence, mevalonate synthesis in extrahepatic tissues. We have shown that mevalonate, the product of HMG-CoA reductase, promotes the growth of breast cancer cells. Because there is no uptake of pravastatin by most extrahepatic cells, this statin will be unable to mitigate the increase in mevalonate synthesis in extrahepatic tissues that accompanies the decrease in circulating cholesterol caused by its inhibition of hepatic HMG-CoA reductase.
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Hydroxymethylglutaryl-CoA reductase
Lovastatin
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