| Project/Area Number |
11680639
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Functional biochemistry
|
|---|
| Research Institution | Jikei University School of Medicine |
|---|
Principal Investigator |
MURAKAMI Yasuko The Jikei University School of Medicine, Biochemistry, professor, 医学部, 教授 (30056709)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MATSUFUJI Senya The Jikei University School of Medicine, Biochemistry, professer, 医学部, 教授 (50192753)
|
|---|
| Project Period (FY) |
1999 – 2001
|
| Project Status |
Completed (Fiscal Year 2001)
|
| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
|
|---|
| Keywords | Antizyme / Antizyme family / Ornithine decuroboxylase / Polyamines / Antizyme inhibitor / Pombe / Zebrafish / Proteasome / オルニチン脱炭酸酵素 / ユビキチン経路 / アンチザイムインヒピター / ノックアウト細胞 / 細胞内蛋白質分解 / ゼブラフィツシュ / ポリアミン輸送 / 分子多様性 / 細胞内タンパク分解 / クローニング |
|---|
| Research Abstract |
Two members of antizymes (AZ) family were cloned from a zebrafish cDNA library together with ODC and AZ inhibitor.The the two AZs (AZS and AZL) were differentially expressed in the tissues from embryos to adult fish. Both AZs inhibited ODC and the inhibition was reversed by AZ inhibitor. AZS but not AZL accelerated ODC degradation in vitro. In a zebrafish cell line, the addition of spermidine caused a rapid degradation of ODC in a protein synthesis-dependent manner, suggesting the presence of the polyamine/AZ-mediated ODC regulatory system. In Schizosaccharomyces pombe, ODC was shown to be rapidly repressed in response to addition of spermidine through AZ-dependent ODC degradation catalyzed by the proteasome. Mammalian AZ 2 did not accelerate ODC degradation in vitro, although AZ2 expression in HTC cells decreased the ODC half-life and inhibited polyamine uptake.The ODC degradation was accelerated by AZ2 itself but not by released AZi by AZ2.In AZiknockout cells derived from knockout mice, spermidine caused rapid ODC degradation in a protein synthesis-dependent manner and AZ 2 was detected in the cell extract. Expression of AZi or 2 in excess caused the growth inhibition in HTC cells. From these results we concluded that AZ 2 negatively regulates polyamine metabolism in mammalian cells. Cellular localization and degradation of AZs were examined. Both AZ1 and AZ2 complexed with ODC were localized in cytosol, whereas free AZs (unbound with ODC) were mainly localized in nucleus. Experimental results with inhibitors suggested that both AZi and AZ2 were degraded in cytosol by the proteasome. Furthermore, in vitro degradation study suggested that both AZs were degraded through the ubiquitin pathway. We also showed the inhibition of ODC degradation in vitro by antizyme 3, tissue specific regulation of mouse kidney ODC and the lack of incorporation of AZi into the 26S proteasome during ODC degradation.
|
