An L- to D-Amino Acid Conversion in an Endosomolytic Analog of the Cell- penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape
An L- to D-Amino Acid Conversion in an Endosomolytic Analog of the Cell- penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape
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DOI：
10.1074/jbc.m116.759837
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发表时间：
2017-01-20
影响因子：
4.8
通讯作者：
Pellois, Jean-Philippe
中科院分区：
文献类型：
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作者：
Najjar, Kristina;Erazo-Oliveras, Alfredo;Pellois, Jean-Philippe
细胞穿透肽（CPP）是公认的细胞不可渗透的货物的递送剂。CPP理论上也可用于调节细胞内过程。然而，它们对蛋白水解降解的敏感性通常限制了它们在这些应用中的效用。先前的研究已经探索了将CPP中的残基从L-立体化学转化为D-立体化学的细胞摄取的后果，但是已经报道了相互矛盾的结果，并且尚未探索细胞内活性途径中的具体步骤。在这里，我们使用二聚体fluorescenceTAT作为模型CPP探索L-到D-立体化学转化的更广泛的后果。我们发现，反转的手性提供蛋白酶抗性，而不改变细胞进入的整体模式，一个过程涉及内吞摄取，然后内体逃逸和胞质访问。然而，尽管手性的反转减少了内吞摄取，但是D-肽一旦进入内体，比其L-对应物更容易逃逸。此外，D-肽在细胞的胞质溶胶中保留数天，而L-肽在数小时内降解。值得注意的是，虽然L-肽对细胞相对无害，但D-肽发挥延长的抗增殖活性。总之，我们的研究结果建立了手性，蛋白酶抗性，细胞渗透性和细胞内活性之间的联系，这可能有助于开发具有改进性能的未来递送剂。
Cell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from L-to D-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescenceTATas a model CPP to explore the broader consequences of L-to D-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the D-peptide, once in the endosome, is significantly more prone to escape than its L-counterpart. Moreover, the D-peptide is retained in the cytosol of cells for several days, whereas the L-peptide is degraded within hours. Notably, while the L-peptide is relatively innocuous to cells, the D-peptide exerts a prolonged antiproliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties.