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-p53-4^Rho. We prepared a panel of SAH-p53-4^Rho variants 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-p53-8^Rho, 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.

我们以前曾报道过，微型蛋白质在折叠的α螺旋（五arg模体）上包含5个精氨酸残基的独特阵列，它们从内体高效地运输到哺乳动物细胞的细胞质和细胞核中。在这里，我们评估五-arg主题是否可以改善细胞内其他不渗透碳氢键合的肽SAH-p53-4 ^Rho的贩运。我们准备了一组SAH-p53-4 ^Rho变体，其中包含具有不同间距和轴向排列的五-arg序列，并评估了它们的整体摄取（通过流式细胞仪判断）和细胞内通路（通过荧光相关光谱法，FCS确定）。该小组的一名成员非常出色地达到了胞质溶胶，与SAH-p53-8 ^Rho达到的水平相当，一种先前报道的高渗透性碳氢化合物固定肽。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确定的胞浆进入之间没有关系。该结果重申，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/或核的进入，必须使用定量和非扩增工具（例如FCS）直接测量浓度。正如对高细胞渗透性微型蛋白（例如ZF5.3）所观察到的，当penta-arg基序位于更多（而不是更少）结构化区域内时，可以使烃类固定肽最佳渗透到细胞质中。