Elucidation of the mechanisms regulating the vascular tone and proliferation : Development of a novel technique to introduce protein into the intact cells and its applications

阐明调节血管张力和增殖的机制：开发将蛋白质引入完整细胞的新技术及其应用

基本信息

批准号：
15590758
负责人：
HIRANO Katsuya
金额：
$ 2.24万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

By using a cell-penetrating peptide, which is found in the human immunodeficiency viral transcription factor Tat and is composed of 11 amino acids, I have developed a novel technique to rapidly, reversibly and quantitatively introduce proteins into the vascular tissues and cells. The cargo protein to be introduced into the cells was prepared as a recombinant protein fused to the cell-penetrating peptide using a bacterial expression system. I have developed the expression vectors. Using this new technique of the protein transduction, I have made the following contribution to the vascular biology.(1)I have for the first time found that the N-terminal region of the regulatory subunit of myosin phosphatase, MYPT1, plays a physiological role in regulating the vascular tone in an intact artery.(2)By introducing the inhibitor proteins of the small G proteins RhoA and Rac1 in a time-specific manner, I have for the first time found that the activity of Rho proteins is required at the late G1 ph … More ase for the cell cycle to progress from the G1 phase to the S phase in the vascular endothelial cells.(3)I found that the 24 h treatment of the arterial tissue with the RhoA inhibitor protein attenuated the arterial contractility in a manner dependent on the endothelial NO production. However, Rac1 inhibitor protein had no such effect. This finding thus for the first time present direct evidence that RhoA plays a physiological role in the production of NO in in situ endothelial cells.(4)I found that the 24 h treatment of the cultured smooth muscle cells with the Rac1 inhibitor protein reduced the cell surface expression of the thrombin receptor PAR1. However, the RhoA inhibitor protein had no such effect. This observation thus suggests that Rac1 plays a critical role in determining the expression of PAR1 in the vascular smooth musclecells.(5)Estrogen inhibited the TNF-α-induced apoptosis in the vascular endothelial cells. However, the introduction of the dominant negative mutant of Akt inhibited the anti-apoptotic effect of estrogen. This finding thus provide the first direct evidence that Akt plays a critical role in the anti-apoptotic signaling elicited by estrogen in the vascular endothelial cells. Less

通过使用细胞渗透的肽，Whooman Imunodficity transtir Transtir Transtir触发者TATs由11个氨基酸组成，开发了一种新的技术，可以快速，可逆性和定量介绍血管组织和细胞引入细胞作为使用轴向体系的植物组合融合到细胞 - 二肾上腺素的肽中。完整的动脉中的血管张力。 e依赖于内皮产生的方式，rac1抑制剂蛋白没有这种作用。 RAC1抑制剂蛋白的细胞降低了RhoA抑制剂蛋白的细胞表面表达，RAC1在确定PAR1在血管平滑肌肉中的表达中起着至关重要的作用。 TNF-α诱导的血管渗透细胞的凋亡引入了雌激素的主要阴性突变体。