| Project/Area Number |
15590331
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Experimental pathology
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
SATOH Hitoshi (2004) The University of Tokyo, Graduate School of Frontier Sciences, Associate Professor, 大学院新領域創成科学研究科, 助教授 (70183829)
森 茂郎 (2003) 東京大学, 医科学研究所, 教授 (30010424)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ASAGIRI Masataka Tokyo Medical and Dental University, Graduate School, Assistant Professor for COE project, 大学院医歯学総合研究科, COE特任助手 (20372435)
ENDO Hideya Totori University, School of Medicine, Emeritus Professor, 医学部, 名誉教授 (40037320)
朝霧 成拳 東京大学, 医科学研究所, 日本学術振興会特別研究員
佐藤 均 東京大学, 医科学研究所, 助手 (70183829)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Project Status |
Completed (Fiscal Year 2004)
|
| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
|
|---|
| Keywords | Vascular endothelial cell / Neovasucularization / Calcium binding protein / Tumor / Metastasis |
|---|
| Research Abstract |
S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. We have revealed that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for protein angiogenesis inhibitors such as fumagillin and ovalicin. By a yeast two-hybird screening system, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays have showed S100A4 associates with MetAP2 in a calcium-dependent manner. The binding site of S100A4 was found to be located within the region between amino acid residures 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 co-localized in both quiescent and basic fibroblast growth factor (bFGF)-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular dist
… More
ribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis.
In order to verify whether the association of two proteins finally influence to form new blood vessels, we first performed an overexpression experiment of the binding domain of MetAP2 in bFGF-stimulated murine vascular endothelial MSS31 cells. Incorporation of BrdU at DNA synthesis phase of the cell cycle was suppressed by overexpression of amino acid residues 1-229 compare to the case of overexpression by amino acid residures 1-169, however, the oscillation rate of intra-cellular calcium ion was maintained as the same level. These results suggest that the activated S100A4 by intracellular calcium influx suppresses DNA synthesis of vascular endothelial cells and may regulate their proliferation through the specific binding to MetAP2. We further prepared four synthesized peptides (p38 ; involving 39 amino acids from 170 to 208 of MetAP2, p39 ; 38aa from 192 to 229, p8 ; 30aa from 175 to 204, and p9 ; 23aa from 170 to 192) to minimize the binding domain to S100A4 in calcium-dependent manner. By using BIACORE system for quantitative measurement of protein-protein interaction, it had shown that p38 has more than twice binding activity to GST-S100A4 compare to 60 as (amino acid residures from 170 to 229 of MetAP2) and p8, but neither of p39 and p9 had binding acitivity. These results indicate that the 23^<rd> or 24^<th> of amino acid residues from N-terminus of MetAP2 is crusial for maintaining the binding activity to S100A4 in calcium-dependent manner.
Our data demonstrate that the possibility of therapoietic suppression of neovascularization in tumor cell growth by treatment of small- sized peptides as a molecular target. Less
|
