Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells
Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells
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DOI：
10.1042/bj20061808
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发表时间：
2007-04-15
影响因子：
4.1
通讯作者：
Jones, Arwyn T.
中科院分区：
文献类型：
--
作者：
Fretz, Marjan M.;Penning, Neal A.;Jones, Arwyn T.
描述细胞穿透肽（如HIV-Tat肽、寡聚腺苷酸和穿透素）进入细胞的机制最近受到了严格的审查。对这些实体的高度兴趣源于它们增强相关大分子（如基因和蛋白质）的细胞递送的能力，这表明它们可能作为药物递送载体具有广泛的应用。提出的摄取机制包括能量非依赖性质膜转位和能量依赖性囊泡摄取和通过内吞途径内化。本文研究了温度、肽浓度和细胞膜胆固醇水平对穿膜肽L-八精氨酸（L-R8）及其D-对映体（D-R8）在CD34（+）白血病细胞中摄取的影响。我们发现，在4 - 12摄氏度，L-R8均匀地标记细胞质和细胞核，但在与D-R8孵育的细胞中，存在核仁的额外标记，其在30摄氏度孵育时仍然突出。在12至30 ℃的温度下，肽也定位于内吞囊泡，因此在37 ℃孵育的细胞中，内吞囊泡作为唯一的标记结构出现。在37 ° C孵育中细胞外肽浓度的小幅增加导致定位于细胞质溶胶并促进D-R8与核仁结合的肽部分的急剧增加。通过用甲基-β-环糊精提取质膜胆固醇，也实现了对细胞质、细胞核和核仁的增强标记。这些数据表明，这些肽的两种温度依赖性摄取机制以及内吞摄取的阈值浓度的存在，当超过阈值浓度时，内吞摄取会促进跨质膜的直接易位。
Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34(+) leukaremia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.