Structure and Regulation of Cholesterol Biosynthesis

以大鼠角鲨烯环氧化酶（SE）和羊毛甾醇合成酶（OSC） cdna为探针，分离了人角鲨烯环氧化酶（SE）和羊毛甾醇合成酶（OSC） cdna。从大鼠和人类基因组文库中也分离到了SE基因。我们发现人类SE基因由11个外显子组成，位于染色体8q端粒附近。研究了甾醇与3-羟基-3-甲基戊二酰辅酶a还原酶（HMG-CoA R）和低密度脂蛋白受体（LDLR）对SE基因表达的调控作用。结果表明，内源性甾醇可以在类似于HMG-CoA R和LDLR的转录水平上调控SE的表达。使用5‘和3’启动子缺失突变体的荧光素酶测定确定了甾醇调节的两个关键区域。Gelshift和过表达实验表明，甾醇调节元件结合蛋白（SREBP）结合到关键区域1，介导SE基因的转录调控。另一个关键区域包含NF-Y结合位点。哺乳动物SE的氨基酸序列显示了5个跨膜结构域，包括FAD结合域的beta_1- α -beta_2基序。酿酒酵母菌、pombe裂糖酵母菌和白色念珠菌则有2个跨膜结构域。这些差异可能反映了SE抑制剂的物种特异性。在DELTA^<100> n端片段（mw 12,000）的两个多肽中发现了底物及其竞争性抑制剂的光亲和标记类似物，分别包括高度保守的序列I和非常接近高度保守的序列II的三肽。结果表明，高度保守的I和II区可能构成了底物结合域的三维结构。

Human squalene eposidase (SE) and lanosterol synthase (OSC) cDNAs were isolated using rat SE and rat OSC cDNAs as probes. SE genes also isolated from rat and human genomic libraries. We found the human SE gene consisted of 11 exons and located in the neighborhood of chromosome 8q telomere. Regulation of SE gene expression by sterol was studied in comparison with those of 3-hydroxy-3-methylglutaryl CoA reductases (HMG-CoA R) and low density lipoprotein receptor (LDLR). Results suggest that sterol produced endogenously can regulate SE expression at the level of transcription similar to HMG-CoA R and LDLR.Luciferase assay using 5'and 3'promoter deletion mutants determined two critical regions for regulation by sterols. Gelshift and overexpression assays showed that sterol regulatory element binding protein (SREBP) binds to critical region l and mediated the transcriptional regulation of SE gene. Another critical region contains NF-Y binding site.The amino acid sequence of mammal SE reveals 5 transmembrane spanning domains including beta_1-alpha A-beta_2 motif of FAD binding domain. In contrast Saccharomyces cerevisiac, Schizosaccharomyces pombe and Candida albicans reveal 2 membrane spanning domains. These differences may reflect species specificity of SE inhibitors. Photoaffinity labeled analogues of substrate and its competitive inhibitors were found in two polypeptides of the DELTA^<100> N-terminal fragment (mw 12,000) including the highly conserved sequence I and the tripeptide very close to the highly conserved II,respectively. The results indicated that the higyly conserved I and II region may constitute three dimensional structure of substrate binding domain.