Evidence for an amphipathicity independent cellular uptake of amphipathic cell-penetrating peptides
Evidence for an amphipathicity independent cellular uptake of amphipathic cell-penetrating peptides
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DOI：
10.1046/j.1432-1327.2000.01681.x
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发表时间：
2000-10-01
期刊：
影响因子：
--
通讯作者：
Oehlke, J
中科院分区：
文献类型：
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作者：
Scheller, A;Wiesner, B;Oehlke, J
用共聚焦激光扫描显微镜（CLSM）结合高效液相色谱（HPLC）研究了一组由膜透性cu-螺旋两亲肽衍生的肽通过结构形成倾向和电荷的逐步改变而获得的细胞摄取。对于CLSM监测，采用了一种在线协议，以避免因冲洗而导致摄取结果的偏差。使用该方案，广泛的荧光，接近外部肽的强度，在所有情况下，在几分钟内观察到细胞质和细胞核，无论两性的程度。细胞裂解物的高效液相色谱分析显示，未代谢的肽是细胞内荧光的主要来源。两性依赖性的显著差异仅在洗涤装载肽的细胞后才显现出来，这反映了两性对洗涤抗性的影响。将细胞暴露于37℃和0℃的多肽中，结果相似，表明了摄取的非内吞特性。从实际应用的角度来看，本研究的结果开启了利用非两亲性肽作为极性化合物转移到细胞内部的载体的可能性，这将绕过目前与使用两亲性载体肽相关的实质性障碍，例如膜毒性和低溶解度。此外，在所研究的多肽系列的几个成员在不同细胞类型中摄取的差异为设计细胞类型特异性载体肽提供了有希望的基础。
The cellular uptake of a peptide set derived from membrane-permeable cu-helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of ampiphathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity-dependent differences became apparent only after washing the peptide-loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 degrees C led to similar results, indicating the nonendocytic character of the uptake.With a view to practical applications, the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell-type specific vector peptides.