David H. Koch Center, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.
Nat Commun. 2012 Apr 17;3:788. doi: 10.1038/ncomms1773.
Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.
噬菌体展示筛选允许在细胞表面研究功能蛋白-蛋白相互作用，但研究细胞内细胞器仍然是一个挑战。在这里，我们引入内化噬菌体文库，以鉴定通过受体非依赖性机制进入哺乳动物细胞并靶向特定细胞器的克隆，作为选择配体肽和鉴定其细胞内受体的工具。我们证明，穿透肽（一种来源于触角足的肽）可以展示在噬菌体包膜上，并介导内化噬菌体通过受体非依赖性途径进入细胞。我们还表明，展示已建立的线粒体特异性定位信号的内化噬菌体构建体靶向线粒体，并且内化噬菌体随机肽文库选择定位于不同细胞内区室的肽基序。作为概念验证，我们证明了这样一种肽，如果化学融合到穿透肽上，就可以受体非依赖性地内化，定位于线粒体，并促进细胞死亡。这种组合平台技术在细胞生物学和药物开发中有潜在的应用。
