Regulatory mechanism of Enzymes of Cholesterol Biosynthesis

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We have studied on structure and regulation aspects of mammalian squalene epoxidase (SE), the late stage of cholesterol biosynthetic enzyme.1. Determination of Functional Domains of SE : Using benzophenone derivatives of FAD,trinorsqualene alcohol and NB-598, we have determined putative binding domains for substrate and NB-598. We also found that terbinafine resistant gene in yeast replaced the only one amino acid compared to wild type yeast (L*F).2. Regulation of SE : Regulation of SE gene expression was studied in comparison with those of HMG-CoA reductase and LDL-receptor using HeLa cells. The results suggest that sterol produced endogenously can regulate SE expression at the level of transcription.3. Structure of human SE genomic DNA : Total length of human SE gene is about 17 kilobase including 11 exons. The introns are ranging from 87 to 3.2 kb. Exon I and II contained putative memmbrane binding domain and FAD-binding followed substrate binding domains, respectively. The identica … More l undecapeptide in common with yeast and mammlaina SE is localized in the exon VIII.4. Chromosomal localization of human SE gene : We have dteremined the regional localization of human SE by using Stanford G3 radiation hybrid panel. Computer analysis of the PCR products for each panel suggests that human SE sequence tagged site is linked with micro-satellite marker D8S508. The marker is localiz-ed q24.13-q telomere of chromosome 8. It is possble that Langer-Giedion syndromeis one of the candidate for SE defect.5. Analyzes of promoter regions of human and rat SE genes : We have determined 5'-sequenec of rat and human SE genes from each genomic clones and aligned 5'-flanking sequences of initiation codons. In both human and rat, the well-conserved region is moticed at the location of 700 or 800 bp upstream from initiation codon. Rat transcriptional initiation site that determined by primer extension method was in 3'-region of the conserved region. To determine the regulatory element of human SE promoter, we preparaed several chimeric lucife-rase plasmids and fusion constructs with deleted human SE promoter and transfected in HeLa cells. The two limited conserved region, a classical SRE-like region and NY-F regions of rat and human may be cruscial for regulation of SE gene by sterols. Less