Department of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057, Zurich, Switzerland.
Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud university medical center, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
Sci Rep. 2017 Oct 16;7(1):13194. doi: 10.1038/s41598-017-13469-y.
Over many years, a variety of delivery systems have been investigated that have the capacity to shuttle macromolecular cargoes, especially proteins, into the cytosol. Due to the lack of an objective way to quantify cytosolic delivery, relative delivery efficiencies of the various transport systems have remained unclear. Here, we demonstrate the use of the biotin ligase assay for a quantitative comparison of protein transport to the cytosol via cell-penetrating peptides, supercharged proteins and bacterial toxins in four different cell lines. The data illustrate large differences in both the total cellular internalization, which denotes any intracellular location including endosomes, and in the cytosolic uptake of the transport systems, with little correlation between the two. Also, we found significant differences between the cell lines. In general, protein transport systems based on cell-penetrating peptides show a modest total uptake, and mostly do not deliver cargo to the cytosol. Systems based on bacterial toxins show a modest receptor-mediated internalization but an efficient delivery to the cytosol. Supercharged proteins, on the contrary, are not receptor-specific and lead to massive total internalization into endosomes, but only low amounts end up in the cytosol.
多年来，人们研究了多种输送系统，这些系统能够将大分子货物（特别是蛋白质）输送到细胞质中。由于缺乏一种客观的方法来定量细胞质输送，因此各种输送系统的相对输送效率仍然不清楚。在这里，我们展示了生物素连接酶测定法在四种不同细胞系中用于定量比较通过穿透细胞肽、超荷蛋白和细菌毒素将蛋白质输送到细胞质的用途。数据说明了总细胞内吞作用（表示包括内体在内的任何细胞内位置）和输送系统的细胞质摄取之间存在很大差异，两者之间几乎没有相关性。此外，我们还发现细胞系之间存在显著差异。一般来说，基于穿透细胞肽的蛋白质输送系统显示出适度的总摄取，并且主要不能将货物输送到细胞质中。基于细菌毒素的系统表现出适度的受体介导内吞作用，但能有效地将货物输送到细胞质中。相反，超荷蛋白不是受体特异性的，会导致大量内吞进入内体，但只有很少的部分进入细胞质。
