Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide
Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide
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DOI：
10.1042/bj20090042
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发表时间：
2009-06-01
影响因子：
4.1
通讯作者：
Jones, Arwyn T.
中科院分区：
文献类型：
--
作者：
Watkins, Catherine L.;Schmaljohann, Dirk;Jones, Arwyn T.
细胞穿透肽（例如寡聚精氨酸和穿透蛋白）穿过生物膜的确切机制尚未阐明，但如果它们想充分发挥作为细胞递送载体的潜力，这是必需的。在本研究中，在许多悬浮和贴壁细胞系中，对温度、肽浓度和质膜胆固醇对模型细胞穿透肽八精氨酸的摄取和亚细胞分布的影响进行了定性和定量分析。当在油冰上进行实验时，2μM细胞外浓度的肽有效地进入并均匀地标记了所研究的所有悬浮细胞的细胞质。但需要高 10 倍的浓度才能在贴壁细胞中观察到类似的结果。在 37 摄氏度和更高的肽浓度下，延时显微镜实验表明，肽快速穿透悬浮细胞的整个质膜，没有证据表明它需要成核区来促进这种效果。用甲基-β-环糊精进行的胆固醇描述增强了在37℃下八精氨酸穿过悬浮细胞质膜的易位，但降低了总肽累积离子。在贴壁细胞中的相同条件下，这对肽的摄取或分布没有影响。胆固醇消耗增加了KG 1a细胞中肽tat 4 C的总体积累，但这种效应可以通过重新添加胆固醇及其甲基-β-环糊精-胆固醇复合物来逆转。结果强调了悬浮细胞质膜对该肽的相对较高的孔隙率，尤其是其低温。表明该功能可用于传递生物活性实体。
The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they tire to reach their full potential as cellular delivery vectors. In the present study, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell-penetrating peptide octa-arginine was performed in a number of suspension and adherent cell lines. When experiments were performed oil ice, the peptide at 2 mu M extracellular concentration efficiently entered and uniformly labelled the cytoplasm oh all the suspension cells studied. but it 10-fold higher concentration was required to observe similar results ill adherent cells. At 37 degrees C and at higher peptide concentrations, time-lapse microscopy experiments showed that the peptide rapidly penetrated the entire plasma membrane of suspension cells, with no evidence of it requirement for nucleation zones to promote this effect. Cholesterol depiction with methyl-beta-cyclodextrin enhanced translocation of octa-arginine across the plasma membrane of suspension cells at 37 degrees C. but decreased overall peptide accumulation ion. Under the same conditions in adherent cells this went had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide tat 4 C in KG la cells, but this effect could be reversed by re-addition of cholesterol its methyl-beta-cyclodextrin-cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially its low temperatures. suggesting thin this feature could be exploited for delivering bioactive entities.