Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, United States.
UNC Eshelman School of Pharmacy and UNC School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
ACS Chem Biol. 2020 Sep 18;15(9):2355-2363. doi: 10.1021/acschembio.0c00319. Epub 2020 Aug 24.
Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.
细胞穿透肽（CPPs）通常用于将大分子递送到活的人类细胞中。为了进入细胞的胞质空间，CPP 通常会使内体的膜通透性增加。反过来，已经开发了几种方法来提高 CPP 的内体膜通透性活性，以提高递药效率。然而，内吞作用途径对于维持细胞内稳态很重要，了解内体通透性如何影响细胞对于定义 CPP 的一般用途至关重要。在此，我们研究了基于 CPP 的递药方案如何影响内吞作用网络。我们发现，在某些情况下，细胞穿透会激活 Chmp1b、Galectin-3 和 TFEB，它们分别是内体修复、细胞器清除和生物发生途径的组成部分。我们还发现，细胞递药会调节内吞作用和内吞作用蛋白水解。值得注意的是，原型 CPP TAT 的多聚体类似物能够有效地使内体通透性增加，而不会诱导膜损伤反应。这些结果挑战了这样一种观点，即使内体渗漏的试剂通常是有毒的。相反，我们的数据表明，有可能以最小的有害影响进入细胞。
