Letoha T, Gaál S, Somlai C, Czajlik A, Perczel A, Penke B
Department of Medical Chemistry, University of Szeged, Szeged, Hungary.
J Mol Recognit. 2003 Sep-Oct;16(5):272-9. doi: 10.1002/jmr.637.
The third helix of the homeodomain of the Antennapedia homeoprotein can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. A 16-amino acid-long peptide fragment, called penetratin, is internalized by the cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, penetratin and two analogs were synthesized:The conformation of penetratin peptides 1-3 was examined in both extracellular matrix-mimetic and membrane-mimetic environments. (1)H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. Peptides 1-3 were labeled by reacting their N-terminal free amino group with fluorescein isothiocyanate (FITC). Membrane translocation of the labelled peptides was studied with cell cultures [WEHI 164 murine fibrosarcoma cells (WC/1); chicken fibroblast cells (CEC-32); chicken monocytic cells (HD-11); human fibroblast (SV 80) and human monocytic cells (MonoMac-6)]. Confocal laser scanning microscopy and flow cytometry assay were used to study membrane translocation. Amphiphilicity was calculated for each peptide. In our experiments all the penetratin peptides penetrated into the cells. Helical conformation and membrane translocation ability showed little correlation: substitution of the two Trp with Phe increased the stability of helical conformation but decreased membrane translocation activity. The results of fluorescence microscopy and flow cytometry show that penetratin can be translocated into the cells by two mechanisms: endocytosis and direct transport through the cell membrane.
触角足同源蛋白的同源结构域的第三个螺旋能够穿过细胞膜进入细胞核，并且可以用作递送寡肽和寡核苷酸的细胞内载体。一种由16个氨基酸组成的肽片段，称为穿膜肽，能以一种特定的、非受体介导的方式被细胞内化。为了更好地理解这种转移机制，合成了穿膜肽及其两种类似物：在模拟细胞外基质和模拟膜的环境中研究了穿膜肽1-3的构象。在不同比例的三氟乙醇/水混合物中进行了（1）H-NMR和圆二色光谱测量。通过使肽1-3的N端游离氨基与异硫氰酸荧光素（FITC）反应对其进行标记。用细胞培养物[WEHI 164小鼠纤维肉瘤细胞（WC/1）；鸡成纤维细胞（CEC-32）；鸡单核细胞（HD-11）；人成纤维细胞（SV 80）和人单核细胞（MonoMac-6）]研究标记肽的膜转运。使用共聚焦激光扫描显微镜和流式细胞术测定法研究膜转运。计算了每种肽的亲水性。在我们的实验中，所有穿膜肽都能进入细胞。螺旋构象与膜转运能力之间几乎没有相关性：用苯丙氨酸取代两个色氨酸增加了螺旋构象的稳定性，但降低了膜转运活性。荧光显微镜和流式细胞术的结果表明，穿膜肽可以通过两种机制转运到细胞中：内吞作用和直接穿过细胞膜的运输。
