Modeling the endosomal escape of cell-penetrating peptides:: Transmembrane pH gradient driven translocation across phospholipid bilayers
Modeling the endosomal escape of cell-penetrating peptides:: Transmembrane pH gradient driven translocation across phospholipid bilayers
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DOI：
10.1021/bi051356w
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发表时间：
2005-11-15
期刊：
影响因子：
2.9
通讯作者：
Gräslund, A
中科院分区：
文献类型：
--
作者：
Magzoub, M;Pramanik, A;Gräslund, A
细胞穿透肽（CPP）能够介导细胞对多种物质的有效摄取。许多 CPP 的内化需要通过内吞作用进行摄取，通过与阴离子细胞表面硫酸乙酰肝素 (HS) 结合启动，然后从内涵体中逸出。为了阐明内体逃逸机制，我们模拟了两种 CPP 的过程：渗透蛋白 (pAntp) 和未加工的牛先验蛋白 (bPrPp) 的 N 端信号肽。产生封装任一肽的大单层磷脂囊泡 (LUV)，并使用离子载体尼日利亚菌素创建跨膜 pH 梯度（Delta pH(mem)，内部呈酸性），类似于体内内体中产生的梯度。在没有 Delta pH(mem) 的情况下，没有观察到 pAntp 从 LUV 中逸出，而有一小部分 bPrPp 逸出。在 Delta pH(mem) 存在的情况下，大量的 pAntp 逃逸，并且发生更高程度的 bPrPp 逃逸。这些结果以及逃逸动力学的差异表明这两种肽的不同逃逸机制。两种肽的逃逸存在最小阈值肽浓度。肽与货物的偶联减少了逃逸部分，而与 H2S 的络合则显着阻碍了逃逸。荧光相关光谱结果表明，在逃逸过程中，LUV 完好无损。总而言之，这些结果提出了 CPP 内体逃逸的模型：Delta pH(mem) 介导的机制，在肽从 HS 解离后，高于最小阈值肽浓度，在不涉及囊泡裂解的过程中。
Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine priori protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (Delta pH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of Delta pH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of Delta pH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: Delta pH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.