Cell-penetrating peptides - A reevaluation of the mechanism of cellular uptake
Cell-penetrating peptides - A reevaluation of the mechanism of cellular uptake
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DOI：
10.1074/jbc.m209548200
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发表时间：
2003-01-03
影响因子：
4.8
通讯作者：
Lebleu, B
中科院分区：
文献类型：
--
作者：
Richard, JP;Melikov, K;Lebleu, B
文献中已将阳离子细胞穿透肽家族（例如 Tat 肽和渗透蛋白）的细胞摄取归因于不涉及内吞作用的机制。在这项工作中，我们重新评估了 Tat 48-60 和 (Arg)(9) 的细胞摄取机制。我们在这里证明，即使在温和的条件下，细胞固定也会导致这些肽的人为摄取。此外，我们表明，除非方案中包括细胞膜吸附肽的胰蛋白酶消化步骤，否则流式细胞术分析不能有效地用于评估细胞摄取。未固定的活细胞的荧光显微镜显示肽的特征性内体分布。流式细胞术分析表明摄取动力学与内吞作用动力学相似。低温孵育和细胞 ATP 池耗尽会抑制肽摄取。对于 Tat 缀合的肽核酸也获得了类似的数据。这些数据与内吞作用参与细胞穿透肽及其与肽核酸的缀合物的细胞内化一致。
Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.