Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu, Tartu, Estonia.
Methods Mol Biol. 2022;2383:229-246. doi: 10.1007/978-1-0716-1752-6_15.
PepFect14 is a cell-penetrating peptide (CPP) derived from stearylated transportan-10 (strearil-TP10) with which it shares the stearic acid residue on C' terminus and the amino acid sequence except for lysines that in PepFect14 are substituted with ornithines. Being non-proteinogenic amino acids, ornithines make PepFect14 less sensitive to serum proteases and due to its positive charges the CPP can form complexes with negatively charged cargos, such as splice correcting oligonucleotides (SCOs), plasmid DNA (pDNA), and proteins. It has been reported that PepFect14/SCO complexes enter the cells mainly through endocytosis, in particular: macopinocitosys and caveolae-mediated endocytosis through the interaction with two receptors of the scavenger receptors class A family (SCARAs). PepFect14 and its complexes trigger the chaperone-mediated autophagy response involving the heat shock protein family (HSP70) whose inhibition leads to an increase of PepFect14 transfection efficacy. Exploiting the interaction between HSP70 and PepFect14 and their ability to form nanoparticle. HSP70 has been delivered in Bomirsky Hamster Melanoma cells (BHM) using PepFect14 of which a protocol is described at the end of this chapter.
PepFect14 是一种细胞穿透肽 (CPP)，源自经过硬脂酰化的转运蛋白 10（strearil-TP10），它与硬脂酸残基共享 C'末端和除赖氨酸以外的氨基酸序列，而赖氨酸在 PepFect14 中被精氨酸取代。由于精氨酸是非蛋白氨基酸，因此 PepFect14 对血清蛋白酶的敏感性降低。由于其正电荷，CPP 可以与带负电荷的货物（如剪接校正寡核苷酸 (SCO)、质粒 DNA (pDNA) 和蛋白质）形成复合物。据报道，PepFect14/SCO 复合物主要通过内吞作用进入细胞，特别是：通过与两个清道夫受体 A 家族 (SCARA) 的受体相互作用，巨胞饮作用和 caveolae 介导的内吞作用。PepFect14 及其复合物引发伴侣介导的自噬反应，涉及热休克蛋白家族 (HSP70)，其抑制导致 PepFect14 转染效率增加。利用 HSP70 与 PepFect14 之间的相互作用及其形成纳米颗粒的能力。HSP70 已使用 PepFect14 递送至博米尔仓鼠黑色素瘤细胞 (BHM)，本章末尾描述了一种方案。
