| Project/Area Number |
11557080
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B).
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
Metabolomics
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
KODAMA Tatsuhiko Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Professor., 先端科学技術研究センター, 教授 (90170266)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KAWABE Yosiki Chugai Pharmaceutical Co., LTD., Senior Scientist., 創薬研究所, 研究員
HAMAKUBO Takao Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Associate Professor., 先端科学技術研究センター, 助教授 (90198797)
川邊 良樹 中外製薬(株), 創薬研究所, 研究員
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥13,500,000 (Direct Cost: ¥13,500,000)
Fiscal Year 2000: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1999: ¥8,900,000 (Direct Cost: ¥8,900,000)
|
|---|
| Keywords | SREBP / protease / cholesterol / SCAP / SSD / S1P / baculovirus / cathepsin B / HMG-CoA還元酵素 / LDL受容体 / HMG-COA還元酵素 / SIP |
|---|
| Research Abstract |
We have been reported that SREBP-2 (sterol regulatory element-binding protein-2) play a key role in the regulation of cholesterol butt not fatty acid metabolism by establishing cell lines in which human SREBP-2 could be induced by IPTG.We also constructed a sensitive assay method for detection of SREBP cutting enzymes using internally quenched fluorogenic peptide substrate, the amino acid sequence of which is the same as the predicted cutting site. By this assay system, we purified neprilysin and cathepsin B as cleaving enzymes in hamster liver microsome fraction. These enzymes were considered to be degradative enzymes for SREBPs in endoplasmic reticulum. The soluble part of human site 1 protease (S1P) was expressed in CHO cells and purified with his-tag affinity column. As the activity of soluble S1P was hardly detectable by the above assay method, we developed a new expression system using the baculovirus. By the baculovirus expression system, we have succeeded to express whole S1P protein and detected Ca^<++> dependent enzymatic activities primarily on S1P expressed budded viruses. We have also detected the expression of membrane proteins such as SREBP-2, SCAP (SREBP cleavage activating protein) and HMG-CoA reductase in viral fractions of culture supernatant of transfected Sf9 cells. This expression system is considered to be a useful tool for expressing membrane proteins. On the other hand, we analyzed genomic structures of S1P, SCAP, and HMG-CoA reductase, and found that exon-shuffling may have occurred in evolutionally step in those proteins that have sterol sensing domain (SSD).
|
