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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671987/
• 关键词: cell-permeable peptide; synthetic peptide; HIV-1 Tat; NF-kappaB; IkappaB; iNOS; transcription factor; RNA-binding peptide; 細胞内導入; ドラッグデリバリー; ペプチド工学; ハイブリダイゼーション; 細胞内情報伝達; 一酸化チッ素合成酵素

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.

已经开发了几种将肽和蛋白质引入哺乳动物细胞的方法，希望了解细胞内信号转出机制并将这些技术应用于治疗用途，其中使用碱性肽作为载体的方法似乎很有前景。 1 Tat 蛋白 (48-60) 和触角足同源域蛋白 (43-58) 已被报道为细胞可渗透肽 我们使用小鼠比较了这些肽的易位活性。通过荧光显微镜观察巨噬细胞RAW 264.7细胞对马来酰亚胺荧光素二乙酸酯标记的肽的细胞摄取和细胞内分布，判断前一种肽具有优于其他相关肽，例如D-取代的类似物和Arg。 Tat 肽的取代类似物也被发现具有细胞渗透性，这意味着这些肽可应用于作为Tat (48-60)在合成肽的细胞内递送中的应用，合成了Tat (48-60)和IκBα(15-49)的杂合肽，其是转录因子的抑制蛋白。 NF-κB 的 N 末端结构域的磷酸化导致 NF-κB 的激活，表明杂合肽在 3 小时内被有效递送到细胞中。还研究了 Tat (48-60) 作为肽载体的其他 Tat 相关肽介导的细胞内递送。

项目成果

二木 史朗: "蛋白質機能解明と機能創出を目指したペプチド合成" 薬学雑誌. 118巻11号. 493-510 (1998)

Shiro Niki：“旨在阐明蛋白质功能和创造功能的肽合成”，Pharmaceutical Journal，第 118 卷，第 11 期。493-510 (1998)

S.Futaki、他: "Peptide Science 1999"Protein Research Foundatiom. 4 (2000)

S.Futaki 等人：“肽科学 1999”蛋白质研究基金会 4 (2000)。

Shiroh Futaki, Tomoki Suzuki, Wakana Ohashi, Takeshi Yagami, Seigo Tanaka, Kunihiro Ueda, Yukio Sugiura: "INTRACELLULAR DELIVERY OF SYNTHETIC PEPTIDES MEDIATED BY TAT-RELATED PEPTIDES"Peptide Science. (in press). (1999)

Shiroh Futaki、Tomoki Suzuki、Wakana Ohashi、Takeshi Yagami、Seigo Tanaka、Kunihiro Ueda、Yukio Sugiura：“由 TAT 相关肽介导的合成肽的细胞内递送”肽科学。

Tachibana ら: "In trace llular Regulation of Macromolecules Using pH-Sensitwe liposomes and Nuclear Localization Signal" Biochem.Biophys Res.Commun. 251巻2号. 538-544 (1998)

Tachibana 等人：“使用 pH-Sensitwe 脂质体和核定位信号进行大分子的痕量调节”Biochem.Biophys Res.Commun. 第 251 卷，第 2. 538-544 期（1998 年）