Efficient translocation of hybrid peptides through cell membrane for the control of transcription

杂合肽通过细胞膜有效易位以控制转录

基本信息

批准号：
10671987
负责人：
FUTAKI Shiroh
金额：
$ 1.73万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Several methods for the introduction of peptides and proteins into mammalian cells have been developed with the hopes of understanding intracellular signal transudation mechanisms and applying these techniques for therapeutic uses. Among them, methods using basic peptides as carriers seem promising. Two peptides derived from HIV-1 Tat protein (48-60) and Antennapedia homeodomain protein (43-58) have been reported as cell permeable peptides. We have compared the translocation activity of these peptides using mouse macrophage RAW 264.7 cells. Cellular uptake and intracellular distribution of the peptides labeled with maleimido fluorescein diacetate were observed by fluorescence microscopy, and the former peptide was judged to have a superior activity. Other related peptides, such as the D-substituted analog and the Arg-substituted analogs of the Tat peptide were also found to be cell-permeable, which implied the applicability of these peptides to the peptide carriers. As an application of Tat (48-60) to the intracellular delivery of synthetic peptides, a hybrid peptide of Tat (48-60) and IκBα(15-49) was synthesized. IκBα is an inhibitory protein of a transcription factor NF-κB. Phosphorylation in its N-terminal domain leads the activation of NF-κB. The hybrid peptide was efficiently delivered into the cells within 3 h, indicative of the usefulness of Tat (48-60) as a peptide carrier. Intracellular delivery mediated by other Tat-related peptides was also investigated.

已经开发了几种将胡椒化和蛋白质引入哺乳动物细胞的方法，以了解细胞内信号渗出机制并将这些技术应用于治疗用途。其中，使用基本辣椒作为载体似乎有希望的方法。源自HIV-1 TAT蛋白（48-60）和触及同源域蛋白（43-58）的两种胡椒体已被报道为可渗透的辣椒。我们使用小鼠巨噬细胞RAW 264.7细胞比较了这些胡椒体的易位活性。通过荧光显微镜观察到用马替米多荧光素二乙酸盐标记的辣椒的细胞摄取和细胞内分布，并认为前一个肽的活性优越。还发现其他相关的肽，例如D-取代的类似物和TAT肽的Arg取代的类似物，这是可渗透的，这暗示这些肽对肽载体的适用性。作为合成肽的细胞内递送的TAT（48-60），合成了TAT（48-60）和IκBα（15-49）的杂化肽。 IκBα是转录因子NF-κB的抑制蛋白。其N末端结构域中的磷酸化导致NF-κB的激活。在3小时内将杂化肽有效地递送到细胞中，这表明TAT（48-60）作为肽载体的有用性。还研究了由其他与TAT相关的Petides介导的细胞内递送。