Thorén Per E G, Persson Daniel, Isakson Petter, Goksör Mattias, Onfelt Agneta, Nordén Bengt
Department of Chemistry and Bioscience, Chalmers University of Technology, Kemivägen 10, SE-412 96, Gothenburg, Sweden.
Biochem Biophys Res Commun. 2003 Jul 18;307(1):100-7. doi: 10.1016/s0006-291x(03)01135-5.
Cell-penetrating peptides are regarded as promising vectors for intracellular delivery of large, hydrophilic molecules, but their mechanism of uptake is poorly understood. Since it has now been demonstrated that the use of cell fixation leads to artifacts in microscopy studies on the cellular uptake of such peptides, much of what has been considered as established facts must be reinvestigated using live (unfixed) cells. In this work, the uptake of analogs of penetratin, Tat(48-60), and heptaarginine in two different cell lines was studied by confocal laser scanning microscopy. For penetratin, an apparently endocytotic uptake was observed, in disagreement with previous studies on fixed cells found in the literature. Substitution of the two tryptophan residues, earlier reported to be essential for cellular uptake, did not alter the uptake characteristics. A heptaarginine peptide, with a tryptophan residue added in the C-terminus, was found to be internalized by cells via an energy-independent, non-endocytotic pathway. Finally, a crucial role for arginine residues in penetratin and Tat(48-60) was demonstrated.
细胞穿透肽被视为用于细胞内递送大分子亲水性分子的有前景的载体，但其摄取机制仍知之甚少。由于现已证明在关于此类肽的细胞摄取的显微镜研究中使用细胞固定会导致假象，因此许多被视为既定事实的内容必须使用活（未固定）细胞重新进行研究。在这项工作中，通过共聚焦激光扫描显微镜研究了穿膜肽、Tat(48 - 60)和七聚精氨酸类似物在两种不同细胞系中的摄取情况。对于穿膜肽，观察到明显的内吞摄取，这与文献中先前对固定细胞的研究结果不一致。先前报道对细胞摄取至关重要的两个色氨酸残基的替换并未改变摄取特性。发现一种在C末端添加了色氨酸残基的七聚精氨酸肽通过能量非依赖的非内吞途径被细胞内化。最后，证明了精氨酸残基在穿膜肽和Tat(48 - 60)中的关键作用。
