Energy-independent translocation of cell-penetrating peptides occurs without formation of pores.: A biophysical study with pep-1
Energy-independent translocation of cell-penetrating peptides occurs without formation of pores.: A biophysical study with pep-1
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DOI：
10.1080/09687860601142936
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Castanho, Miguel A. R. B.
中科院分区：
文献类型：
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作者：
Henriques, Sonia Troeira;Quintas, Alexandre;Castanho, Miguel A. R. B.
PEP-1是一种细胞穿透肽(CPP)，具有跨生物膜转运和将活性蛋白引入细胞内的能力。到目前为止，这种CPP使用的摄取机制还不清楚。以前的结果表明，这种机制是非内吞作用的，并表明多肽和脂双层之间的物理化学相互作用控制着转位机制。跨膜孔的形成已被提出，但这个问题一直存在争议。在这项工作中，用CD和ATR-FTIR光谱测定了没有/存在脂类双层时PEP-1的二级结构，并通过平面脂膜的电生理测量和使用巨大的单层囊泡的共聚焦显微镜评价了孔的形成。尽管PEP-1疏水结构域倾向于在脂类双层存在的情况下形成两亲性的α-螺旋构象，但在PEP-1存在的情况下没有证据表明存在膜孔。此外，只有在高肽/脂比的情况下，膜通透性才会发生变化，从而导致膜的完全解体。这些观察表明，在PEP-1与膜的相互作用中，静电相互作用是第一重要的，并表明没有形成孔。在多肽分配过程中可能形成了一种多肽-脂结构，这有利于多肽的转运。
Pep-1 is a cell-penetrating peptide (CPP) with the ability to translocate across biological membranes and introduce active proteins inside cells. The uptake mechanism used by this CPP is, as yet, unknown in detail. Previous results show that such a mechanism is endocytosis-independent and suggests that physical-chemical interactions between the peptide and lipid bilayers govern the translocation mechanism. Formation of a transmembrane pore has been proposed but this issue has always remained controversial. In this work the secondary structure of pep-1 in the absence/presence of lipidic bilayers was determined by CD and ATR-FTIR spectroscopies and the occurrence of pore formation was evaluated through electrophysiological measurements with planar lipid membranes and by confocal microscopy using giant unilamellar vesicles. Despite pep-1 hydrophobic domain tendency for amphipathic alpha-helix conformation in the presence of lipidic bilayers, there was no evidence for membrane pores in the presence of pep-1. Furthermore, alterations in membrane permeability only occurred for high peptide/lipid ratios, which induced the complete membrane disintegration. Such observations indicate that electrostatic interactions are of first importance in the pep-1-membrane interactions and show that pores are not formed. A peptide-lipid structure is probably formed during peptide partition, which favours peptide translocation.