Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
J Biol Chem. 2011 Oct 21;286(42):36932-43. doi: 10.1074/jbc.M111.264424. Epub 2011 Aug 26.
Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.
细胞穿透肽能够穿过活细胞的质膜，因此在药物传递应用中具有潜在的用途。富含二硫键的环肽在药物设计中也有很大的前景，因为它们具有异常的稳定性，但迄今为止，只有一种环肽被报道能够穿透细胞，即苦瓜胰蛋白酶抑制剂 II（MCoTI-II）。MCoTI-II 属于植物来源的环肽家族，其特征是具有环半胱氨酸结基序。先前在固定细胞中的研究表明，MCoTI-II 可以穿透细胞，但来自另一个环肽亚家族的原型环肽 kalata B1 与质膜结合，并且没有转运到细胞内。在这里，我们通过活细胞成像表明，MCoTI-II 和 kalata B1 都可以进入细胞。kalata B1 与 MCoTI-II 具有相同的环半胱氨酸结结构基序，但在序列上有很大的不同，这两种肽进入细胞的机制也不同。MCoTI-II 似乎通过巨胞饮作用进入细胞，可能是通过与细胞膜中的磷酸肌醇相互作用介导的，而 kalata B1 通过靶向磷脂酰乙醇胺磷脂与膜直接相互作用，可能导致膜弯曲和囊泡形成。我们还表明，另一种植物来源的环肽 SFTI-1 也可以穿透细胞。SFTI-1 仅包含 14 个氨基酸，除了其环状骨架外，在结构上与环肽非常不同，环肽的大小是其两倍。有趣的是，SFTI-1 与测试的任何一种磷脂都没有相互作用，其穿透机制似乎与 MCoTI-II 和 kalata B1 不同。不同的富含二硫键的环肽穿透细胞的能力增强了它们在药物设计中的潜力，我们提出了一个新的分类，即环状细胞穿透肽。
