Liao Xiaoli, Rabideau Amy E, Pentelute Bradley L
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue 18-596, Cambridge, MA 02193 (USA).
Chembiochem. 2014 Nov 3;15(16):2458-66. doi: 10.1002/cbic.201402290. Epub 2014 Sep 22.
Antibody mimics have significant scientific and therapeutic utility for the disruption of protein-protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein-protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.
抗体模拟物对于破坏细胞内蛋白质-蛋白质相互作用具有重要的科学和治疗用途；然而，将其递送至细胞溶质仍然是一项重大挑战。在此我们表明，炭疽毒素的一个组分——保护性抗原（PA），当与致死因子（LF）的N端结构域偶联时，能够有效地将常用的抗体模拟物转运至哺乳动物细胞的细胞质中。相比之下，一种细胞穿透肽（CPP）无法将这些抗体模拟物中的任何一种递送至细胞溶质中。通过免疫共沉淀证实了在慢性髓性白血病细胞中，一种转运的串联单克隆抗体与其蛋白质靶点Bcr-Abl的重折叠和结合。我们还观察到该单克隆抗体对Bcr-Abl激酶活性的抑制以及诱导的细胞凋亡。在另一个实例中，我们展示了在PA介导递送靶向hRaf-1的亲合体结合剂后，MAPK信号通路中关键相互作用的破坏。我们首次表明PA能够递送具有生物活性的抗体模拟物以破坏细胞内蛋白质-蛋白质相互作用。这项技术为将这些现代药物的应用扩展至细胞内环境增添了一种有用的工具。
