1993 Fiscal Year Final Research Report Summary
REGULATION OF SQUALENE EPOXIDASE EXPRESSION AND THE ENZYME EVOLUTION
| Project/Area Number |
04454155
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Niigata University |
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Principal Investigator |
ONO Teruo NIIGATA UNIVERSITY SCHOOL OF MEDICINE PROFESSOR, 医学部, 教授 (00000927)
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| Co-Investigator(Kenkyū-buntansha) |
HITOMI Masahiro NIIGATA UNIVERSITY SCHOOL OF MEDICINE RESEARCH ASSOCIATE, 医学部, 助手 (40218730)
FUJII Hiroshi NIIGATA UNIVERSITY SCHOOL OF MEDICINE ASSOCIATE PROFESSOR, 医学部, 助教授 (90165340)
SAKAKIBARA Jun NIIGATA UNIVERSITY SCHOOL OF MEDICINE RESEARCH ASSOCIATE, 医学部, 助手 (90242403)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Squalene Epoxidase / Sterol Regulatory Element(SRE) / Cholesterol / Monooxygenase / Flavoenzyme / Rat |
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| Research Abstract |
We have succeeded in a cloneing of rat squalene epoxidase(RSE).This is the first presentation of mamalian SE.RSE polynucleotide deduced from nucleotide sequence ontains 573 amino acids(Mr. = 63,950 Da).The amino acid sequence reveals one potential transmembrane domain with a hydrophobic segment (Leu^<27> to Tyr^<43>) in the N-terminal region and beta_1-alphaA-beta_2 motif which is known to be the consensus for FAD binding domain. Thereis a moderate homology between the rat and the ERG1 gene encoding SE cloned from an allylamine-resistant Saccharomyces cerevisiae mutant. Expression of a full length RSE protein in Escherichia coli confirms this polypeptide is functional one.
To prove RSE as an important rate llimiting enzyme of cholesterol biosynthetic pathway, we analayzed the cholesterol induced change of RSE activity, protein quantity and mRNA levels. Western blot analylsis revealed that lipoprotein deficient serum caused the induction of RSE in L929 cells. Northern bolt analysis supported that the induction occurred in the transcriptional level. In addition, we found the SRE(Sterol regulatory element)-like sequences in promoter region of the RSE gene. These results strongly suggest that RSE is regulated by cholesterol similar to the rate limiting enzyme, HMG-CoA reductase.
A homology search in the GenBank data base indicated that the cDNA for RSE shows moderate nucleotide sequence similarity to allylamine-resistant Saccharomyces cerevisiae SE.We could not find any similarity to peroxidases as well as cyclooxygenase, lipooxygenases and cytochrome P-450 isozymes. Thus, the RSE may evolved from the distinct ancestral gene.
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