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

为了了解细胞内的信号传导机制，并将这些技术应用于治疗用途，已经开发了几种将多肽和蛋白质引入哺乳动物细胞的方法。其中，使用碱性多肽作为载体的方法似乎很有前途。两种来源于HIV-1Tat蛋白(48-60)和触角线虫同源结构域蛋白(43-58)的多肽已被报道为细胞通透性多肽。我们用小鼠巨噬细胞RAW 264.7细胞比较了这些多肽的易位活性。荧光显微镜下观察了马来酰亚胺荧光素二乙酸酯标记的多肽的细胞摄取和细胞内分布，确定前者具有更好的活性。其他相关的多肽，如TAT的D-取代类似物和Arg-取代类似物也被发现是细胞通透性的，这意味着这些多肽对多肽载体的适用性。作为TAT(48-60)在合成肽细胞内递送方面的应用，合成了TAT(48-60)和I-κBα(15-49)的杂交肽。I-κBα是转录因子NF-κB的抑制蛋白，其N-末端的磷酸化导致核因子-κB的激活，杂交肽在3h内被有效地输送到细胞内，这表明TAT(48-60)作为多肽载体是有用的。对其他TAT相关多肽介导的细胞内给药也进行了研究。

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.