Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
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DOI：
10.1016/j.bbamem.2009.09.014
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发表时间：
2009-12-01
影响因子：
3.4
通讯作者：
Langel, Ulo
中科院分区：
文献类型：
--
作者：
Guterstam, Peter;Madani, Fatemeh;Langel, Ulo
细胞穿透肽（Cell-penetrating peptides，CPP）是一种膜渗透性载体，具有进入细胞内部的内在能力。疏水性抗衡阴离子芘丁酸盐增强寡精氨酸CPP的细胞摄取。为了阐明CPP的吸收机制，研究了芘丁酸盐对公认的具有不同疏水性和精氨酸含量的CPP的影响。通过荧光激活细胞分选、共聚焦显微镜和基于细胞的剪接转换测定来分析细胞CPP摄取和CPP介导的寡核苷酸递送。剪接转换寡核苷酸是2 '-O-甲基RNA和锁核酸的混合物，以非共价复合物形式递送，CPP过量10倍摩尔。在钙黄绿素释放实验中研究了CPP诱导的大单层囊泡膜扰动。我们观察到，芘丁酸促进细胞摄取和转运的寡聚精氨酸九聚体介导的寡核苷酸，而有限的影响芘丁酸更疏水性的CPP观察。通过结合不同的实验结果，我们得出结论，细胞摄取寡精氨酸的途径是由直接膜易位为主，而寡精氨酸介导的寡核苷酸易位的途径是由内吞为主。这两种机制都促进了芘丁酸，我们建议芘丁酸有不同的网站的行动的两个吸收和转运机制。(C)2009 Elsevier B. V.保留所有权利。
Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with varying hydrophobicity and arginine content is investigated. The cellular CPP uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell-based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2'-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms. (C) 2009 Elsevier B.V. All rights reserved.