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首页> 外文期刊>Biochemistry >Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores
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Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores

机译：通过跨脂质膜及其诱导的毛孔易位的细胞穿透肽转运蛋白10进入单个囊泡。

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摘要

The cell-penetrating peptide, transportan 10 (TP10), can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargo such as proteins. However, the mechanisms underlying its translocation and the delivery of large cargo remain unclear. In this report we investigated the entry of TP10 into a single giant unilamellar vesicle (GUV) and the TP10-induced leakage of fluorescent probes using the single GUV method. GUVs of 20% dioleoylphosphatidylglycerol (DOPG)/80% dioleoylphosphatidylcholine (DOPC) were prepared, and they contained a water-soluble fluorescent dye, Alexa Fluor 647 hydrazide (AF647), and smaller vesicles composed of 20% DOPG/80% DOPC. The interaction of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) with these loaded GUVs was investigated using confocal microscopy. The fluorescence intensity of the GUV membrane increased with time to a saturated value, then the fluorescence intensity due to the membranes of the smaller vesicles inside the GUV increased prior to leakage of AF647. This result indicates that CF-TP10 entered the GUV from the outside by translocating across the lipid membrane before CF-TP10-induced pore formation. The rate constant of TP10- induced pore formation in lipid membranes increased with an increase in TP10 concentration. Large molecules such as Texas Red Dextran 40 000, and vesicles with a diameter of 1?2 μm, permeated through the TP10-induced pores or local rupture in the lipid membrane. These results provide the first direct experimental evidence that TP10 can deliver large cargo through lipid membranes, without the need for special transport mechanisms such as those found in cells.
机译：细胞穿透肽transportan 10（TP10）可以跨活细胞的质膜转运，因此可以用于细胞内递送生物货物，例如蛋白质。但是，其易位和大件货物交付的机制尚不清楚。在本报告中，我们调查了TP10进入单个巨大单层囊泡（GUV）的情况，以及使用单个GUV方法检测TP10诱导的荧光探针泄漏。制备了20％的脂酰磷脂酰甘油（DOPG）/ 80％的脂酰磷脂酰胆碱（DOPC）的GUV，它们包含水溶性荧光染料Alexa Fluor 647酰肼（AF647）和由20％DOPG / 80％DOPC组成的较小囊泡。使用共聚焦显微镜研究了羧基荧光素（CF）标记的TP10（CF-TP10）与这些负载的GUV的相互作用。 GUV膜的荧光强度随时间增加至饱和值，然后在AF647泄漏之前，由于GUV内较小囊泡膜的荧光强度增加。该结果表明，CF-TP10通过在CF-TP10诱导的孔形成之前穿过脂质膜移位而从外部进入了GUV。 TP10诱导的脂质膜孔形成速率常数随TP10浓度的增加而增加。大分子（例如得克萨斯红葡聚糖40 000）和直径为1？2μm的囊泡穿过TP10诱导的孔或脂质膜的局部破裂。这些结果提供了第一个直接的实验证据，表明TP10可以通过脂质膜递送大量货物，而无需诸如细胞中发现的特殊转运机制。

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