Publications

• Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol delta8-isomerase, a cholesterogenic enzyme with multiple functions.

  Bae S., Seong J., Paik Y.

  Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Recently, mutations of SI have been found to be associated with Conradi-Hüne ... >> More

  Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Recently, mutations of SI have been found to be associated with Conradi-Hünermann syndrome in humans. To investigate the in vitro and in vivo modes of molecular regulation of SI and its role in cholesterol biosynthesis in mammals, we isolated a full-length cDNA encoding rat SI. The deduced amino-acid sequence of rat SI predicts a 230-residue protein (26737 Da) with 87% and 80% amino-acid identity to mouse and human counterparts. The rat SI gene was mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FISH). The biological function of the cloned rat SI cDNA was verified by overexpressing recombinant Myc-SI in Saccharomyces cerevisiae. It showed a characteristic pattern of inhibition on exposure to trans-2-[4-(1,2-diphenylbuten-1-yl)phenoxy]-N,N-dimethylethylamine (tamoxifen; IC(50)=11.2 microM) and 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A; IC(50)=4.2 microM), two well known potent inhibitors of SI. Northern-blot analysis of 3-week-old rats compared with 2-year-old rats showed that SI mRNA expression in both age groups was restricted to liver, where a 70% reduction in mRNA levels was observed in 2-year-old rats. The FISH studies revealed ubiquitous expression of SI mRNA in rat hepatocytes. The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells. Treatment of H4IIE cells grown in medium supplemented with fetal bovine serum with tamoxifen for 24 h resulted in a dose-dependent induction of SI mRNA, with a concomitant suppression of sterol regulatory element binding protein-1 mRNA. Interestingly, this effect was not seen in emopamil-treated cells. The in vivo experiments also indicate that both mRNA expression and enzymic activity of SI in liver were induced approx. 3-fold in rats fed 5% (w/w) cholestyramine plus 0.1% (w/w) lovastatin in normal chow for 2 weeks. With this newly cloned rat SI cDNA, it becomes possible to gain molecular understanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic diseases. << Less

  Biochem. J. 353:689-699(2001) [PubMed] [EuropePMC]

• A comprehensive machine-readable view of the mammalian cholesterol biosynthesis pathway.

  Mazein A., Watterson S., Hsieh W.Y., Griffiths W.J., Ghazal P.

  Cholesterol biosynthesis serves as a central metabolic hub for numerous biological processes in health and disease. A detailed, integrative single-view description of how the cholesterol pathway is structured and how it interacts with other pathway systems is lacking in the existing literature. He ... >> More

  Cholesterol biosynthesis serves as a central metabolic hub for numerous biological processes in health and disease. A detailed, integrative single-view description of how the cholesterol pathway is structured and how it interacts with other pathway systems is lacking in the existing literature. Here we provide a systematic review of the existing literature and present a detailed pathway diagram that describes the cholesterol biosynthesis pathway (the mevalonate, the Kandutch-Russell and the Bloch pathway) and shunt pathway that leads to 24(S),25-epoxycholesterol synthesis. The diagram has been produced using the Systems Biology Graphical Notation (SBGN) and is available in the SBGN-ML format, a human readable and machine semantically parsable open community file format. << Less

  Biochem. Pharmacol. 86:56-66(2013) [PubMed] [EuropePMC]

  This publication is cited by 30 other entries.

• Purification, characterization and catalytic properties of human sterol 8-isomerase.

  Nes W.D., Zhou W., Dennis A.L., Li H., Jia Z., Keith R.A., Piser T.M., Furlong S.T.

  CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an ... >> More

  CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM) <ketoconazole (156 microM)<cholesterol (620 microM). The results were consistent with stereoelectronic features of isomerization and support the general model for Delta(7)-sterol formation in cholesterol synthesis. << Less

  Biochem J 367:587-599(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Emopamil-binding protein, a mammalian protein that binds a series of structurally diverse neuroprotective agents, exhibits delta8-delta7 sterol isomerase activity in yeast.

  Silve S., Dupuy P.H., Labit-Lebouteiller C., Kaghad M., Chalon P., Rahier A., Taton M., Lupker J., Shire D., Loison G.

  Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers. No sequence data for high eukaryote sterol isomerase being ... >> More

  Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert delta8-sterol into delta7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and delta8-delta7 sterol isomerase are identical proteins in mammals. << Less

  J. Biol. Chem. 271:22434-22440(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin.

  Rahier A., Pierre S., Riveill G., Karst F.

  A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed Zm8,7SI, has been isolated from an EST (expressed sequence tag) library and subcloned into the yeast erg2 mutant lacking 8,7SI activity. Zm8,7SI restored endogenous ergosterol synthesis. An in vitro enzymatic assay in the corresponding ... >> More

  A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed Zm8,7SI, has been isolated from an EST (expressed sequence tag) library and subcloned into the yeast erg2 mutant lacking 8,7SI activity. Zm8,7SI restored endogenous ergosterol synthesis. An in vitro enzymatic assay in the corresponding yeast microsomal extract indicated that the preferred Delta(8)-sterol substrate possesses a single C4alpha methyl group, in contrast with 8,7SIs from animals and fungi, thus reflecting the diversity in the structure of their active site in relation to the distinct sterol biosynthetic pathways. In accordance with the proposed catalytic mechanism, a series of lipophilic ammonium-ion-containing derivatives possessing a variety of structures and biological properties, potently inhibited the Zm8,7SI in vitro. To evaluate the importance of a series of conserved acidic and tryptophan residues which could be involved in the Zm8,7SI catalytic mechanism, 20 mutants of Zm8,7SI were constructed as well as a number of corresponding mutants of the Saccharomyces cerevisiae 8,7SI. The mutated isomerases were assayed in vivo by sterol analysis and quantification of Delta(5,7)-sterols and directly in vitro by examination of the activities of the recombinant Zm8,7SI mutants. These studies have identified His(74), Glu(78), Asp(107), Glu(121), Trp(66) and Trp(193) that are required for Zm8,7SI activity and show that binding of the enzyme-substrate complex is impaired in the mutant T124I. They underline the functional homology between the plant and animal 8,7SIs on one hand, in contrast with the yeast 8,7SI on the other hand, in accordance with their molecular diversity and distinct mechanisms. << Less

  Biochem J 414:247-259(2008) [PubMed] [EuropePMC]