Unique arginine array improves cytosolic localization of hydrocarbon-stapled peptides.
Unique arginine array improves cytosolic localization of hydrocarbon-stapled peptides.
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DOI：
10.1016/j.bmc.2017.11.008
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发表时间：
2017-11
影响因子：
3.5
通讯作者：
Kim Quach;Jonathan Larochelle;Xiao-han Li;E. Rhoades;A. Schepartz
中科院分区：
文献类型：
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作者：
Kim Quach;Jonathan Larochelle;Xiao-han Li;E. Rhoades;A. Schepartz
我们以前曾报道，微型蛋白质含有一个独特的阵列的5个精氨酸残基上折叠的α-螺旋-一个penta-arg基序-交通与高效率从内涵体进入细胞质和细胞核的哺乳动物细胞。在这里，我们评估是否五精氨酸基序可以提高细胞内的运输，否则不渗透的烃钉肽，SAH-p53- 4 Rho。我们制备了一组SAH-p53- 4 Rho变体，其含有具有不同间距和轴向排列的penta-arg序列，并评估了它们的总体摄取（如通过流式细胞术判断的）和它们的细胞内进入（如通过荧光相关光谱法，FCS测定的）。该组中的一个成员非常好地到达胞质溶胶，与SAH-p53- 8 Rho（一种先前报道的高渗透性烃钉合肽）所达到的水平相匹配。值得注意的是，我们发现通过流式细胞术判断的细胞摄取与通过FCS确定的胞质进入之间没有关系。这一结果重申，总体摄取和内体释放代表了根本不同的生物学过程。为了确定胞质和/或核进入，必须使用定量和非扩增工具（如FCS）直接测量浓度。正如已经观察到的高度细胞渗透的微型蛋白质，如ZF 5.3，最佳的渗透烃钉合肽进入细胞胞质溶胶的结果时，五-精氨酸基序位于更多（而不是更少）的结构化区域。
We have previously reported that miniature proteins containing a distinct array of 5 arginine residues on a folded α-helix – a penta-arg motif – traffic with high efficiency from endosomes into the cytosol and nucleus of mammalian cells. Here we evaluate whether a penta-arg motif can improve the intracellular trafficking of an otherwise impermeant hydrocarbon-stapled peptide, SAH-p53-4Rho. We prepared a panel of SAH-p53-4Rhovariants containing penta-arg sequences with different spacings and axial arrangement and evaluated their overall uptake (as judged by flow cytometry) and their intracellular access (as determined by fluorescence correlation spectroscopy, FCS). One member of this panel reached the cytosol extremely well, matching the level achieved by SAH-p53-8Rho, a previously reported and highly permeant hydrocarbon-stapled peptide. Notably, we found no relationship between cellular uptake as judged by flow cytometry and cytosolic access as determined by FCS. This result reiterates that overall uptake and endosomal release represent fundamentally different biological processes. To determine cytosolic and/or nuclear access, one must measure concentration directly using a quantitative and non-amplified tool such as FCS. As has been observed for highly cell permeant miniature proteins such as ZF5.3, optimal penetration of hydrocarbon-stapled peptides into the cell cytosol results when the penta-arg motif is located within more (as opposed to less) structured regions.