Discovery and Characterization of a Peptide That Enhances Endosomal Escape of Delivered Proteins in Vitro and in Vivo
Discovery and Characterization of a Peptide That Enhances Endosomal Escape of Delivered Proteins in Vitro and in Vivo
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DOI：
10.1021/jacs.5b05694
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发表时间：
2015-11-11
影响因子：
15
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Li, Margie;Tao, Yong;Liu, David R.
蛋白质向哺乳动物细胞的低效输送仍然是实现许多蛋白质治疗潜力的主要障碍。我们和其他人之前已经证明，超正电荷的蛋白质可以有效地内吞，并可以将相关的蛋白质和核酸带入细胞。然而，以这种方式运送的绝大多数货物仍留在内体中，不会到达胞浆。在这项研究中，我们设计并实现了一种筛选，以发现能够增强与超正电荷GFP(+36GFP)融合的蛋白的内体逃逸的多肽。从先前报道的破坏微生物膜的多肽中，我们发现了一种由13个残基组成的多肽，Aurein 1.2，在培养细胞的胞质分级试验中，它能显著增加胞浆蛋白的输送，最多可增加5倍。另外四项独立的非内体蛋白递送试验共同表明，金蛋白1.2增强了相关内吞蛋白货物的内体逃逸。结构功能研究阐明了多肽序列和蛋白质结合对内体逃逸活性的要求。当将+36 GFP-Cre重组酶融合基因应用于活体小鼠内耳的体内递送时，与Arein 1.2的融合显著提高了非内体Cre重组酶递送效力，导致高达100%的重组内毛细胞和96%的重组外毛细胞，而没有Arein 1.2的+36-GFP-Cre重组毛细胞的重组毛细胞比例为0-4%。总而言之，这些发现描述了一种遗传上可编码的内容体逃逸增强肽，它可以显著增加阳离子蛋白在体外和体内的细胞质输送。
The inefficient delivery of proteins into mammalian cells remains a major barrier to realizing the therapeutic potential of many proteins. We and others have previously shown that superpositively charged proteins are efficiently endocytosed and can bring associated proteins and nucleic acids into cells. The vast majority of cargo delivered in this manner, however, remains in endosomes and does not reach the cytosol. In this study we designed and implemented a screen to discover peptides that enhance the endosomal escape of proteins fused to superpositively charged GFP (+36 GFP). From a screen of peptides previously reported to disrupt microbial membranes without known mammalian cell toxicity, we discovered a 13-residue peptide, aurein 1.2, that substantially increases cytosolic protein delivery by up to similar to 5-fold in a cytosolic fractionation assay in cultured cells. Four additional independent assays for nonendosomal protein delivery collectively suggest that aurein 1.2 enhances endosomal escape of associated endocytosed protein cargo. Structure function studies clarified peptide sequence and protein conjugation requirements for endosomal escape activity. When applied to the in vivo delivery of +36 GFP-Cre recombinase fusions into the inner ear of live mice, fusion with aurein 1.2 dramatically increased nonendosomal Cre recombinase delivery potency, resulting in up to 100% recombined inner hair cells and 96% recombined outer hair cells, compared to 0-4% recombined hair cells from +36-GFP-Cre without aurein 1.2. Collectively, these findings describe a genetically encodable, endosome escape-enhancing peptide that can substantially increase the cytoplasmic delivery of cationic proteins in vitro and in vivo.