Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans
Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans
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DOI：
10.1021/bc700289w
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发表时间：
2008-03-01
影响因子：
4.7
通讯作者：
Futaki, Shiroh
中科院分区：
文献类型：
--
作者：
Kosuge, Michie;Takeuchi, Toshihide;Futaki, Shiroh
富含精氨酸的细胞穿透肽进入细胞的确切机制仍然是争论的主题。在这里，我们详细分析了血清和细胞外浓度对低聚精氨酸(Rn；n=4，8，12，16)内化的影响。培养液中血清的存在对R12和R16多肽的摄取有较大影响，但对R4和R8的摄取没有明显影响。细胞在37℃与R12和R16肽在含血清的培养液中孵育，显示大多数标记局限于点状内吞结构。在无血清介质中进行相同的实验会导致胞浆标记的急剧增加，在4℃的多肽处理的细胞中也观察到类似的弥漫R12和R16标记。这表明，在这两种情况下，多肽都是通过非内吞机制进入的。对R12肽的进一步研究表明，非内吞摄取和胞浆标记的启动也依赖于血清浓度和胞外肽浓度。在相对较低的浓度下，多肽标记内吞结构，但随着多肽浓度的增加，标记胞浆的比例急剧增加，这伴随着细胞总荧光的非线性增加。膜相关蛋白多糖也有助于增加细胞表面的多肽浓度，通过静电相互作用增强它们的募集。这些结果表明，这些化合物的摄取机制高度依赖于血清的存在和有效的胞外肽浓度。
The exact mechanisms by which arginine-rich cell-penetrating peptides enter cells are still the subject of debate. Here, we have analyzed in detail the effects of serum and extracellular concentration on the internalization of oligoarginines (Rn; n = 4, 8, 12, 16). The presence of serum in the incubation medium had a major influence on the uptake of R12 and R16 peptides but did not affect the uptake of R4 and R8 significantly. Incubation of cells at 37 degrees C with R12 and R16 peptides in serum-containing medium showed that the majority of labeling was confined to punctate endocytic structures. Performing the same experiments in serum-free media led to a dramatic increase in cytosolic labeling, and similarly diffuse R12 and R16 labeling was observed in cells treated with peptides at 4 degrees C. This suggests, in both cases, that the peptides were entering via a nonendocytic mechanism. Further studies on R12 peptide suggest that the initiation of nonendocytic uptake and cytosolic labeling is also dependent on serum concentration and extracellular peptide concentration. At relatively low concentrations, the peptide labels endocytic structures, but upon raising the peptide concentration, the fraction labeling the cytosol increases dramatically and this accompanies a nonlinear increase in total cellular fluorescence. Membrane-associated proteoglycans also contribute to increasing the peptide concentration at the cell surface by enhancing their recruitment via electrostatic interactions. These results demonstrate that uptake mechanisms of these compounds are highly dependent on both the presence of serum and the effective extracellular peptide concentration.