Delivery of protein biosensors across the plasma membrane of live cells

项目摘要 我们的长期目标是建立一种有效地将蛋白质输送到肝细胞胞浆的方法学。 哺乳动物细胞。目前的递送系统，如细胞穿透肽(CPPs)，效率低下，因为 它们促进广泛的内体包裹和蛋白质货物的降解。这将导致 使蛋白质生物传感器的正确成像变得不切实际的实验人工制品。我们建议解决 这一问题是通过优化CPP选择性地破坏内膜的能力来实现的 更有效地将蛋白质从内体释放到胞浆中，并减少降解。我们的特定 目的是：1)确定CPP介导的最佳蛋白质传递所需的条件；2)评估和 优化新型CPP系统，旨在根据管腔的酸化有效地破坏膜 内体，3)确定内体pH、CPP浓度和内体释放活性之间的关系 CPP的数量。为了实现这些目标，我们将使用最近开发的成像技术来明确地 测量进入活细胞的蛋白质探针的胞内和胞浆分布。新蛋白质 可以报告含有CPP-蛋白质结合物的内涵体性质的探针也将被 发展起来的。我们预计，我们的研究结果将在内吞关键步骤中提供关键的化学见解。 并为合理设计高效的交付系统奠定坚实的基础 非靶向蛋白质背景低的蛋白质生物传感器的胞浆靶向。这不仅将使 使用外部给药成像探针对活细胞的显微镜检查，但应该有重要的影响 一般情况下，在整个细胞不渗透大分子的输送领域。

Project Abstract Our long-term goal is to establish a methodology to efficiently deliver proteins to the cytosol of live mammalian cells. Current delivery systems such as cell-penetrating peptides (CPPs) are inefficient because they promote extensive endosomal entrapment and degradation of their protein cargo. This results in experimental artifacts that render the proper imaging of protein biosensors impractical. We propose to solve this problem by optimizing the ability of CPPs to selectively disrupt endosomal membranes so as to achieve a more efficient release of the protein from endosomes into the cytosol and reduce degradation. Our specific aims are to: 1) identify the conditions required for optimal CPP-mediated protein delivery, 2) evaluate and optimize novel CPP systems designed to efficiently disrupt membranes upon acidification of the lumen of endosomes, 3) define the relations between endosomal pH, CPP concentration and endosomal release activity of CPPs. To achieve these goals, we will use a recently developed imaging technique to unambiguously measure the endocytic and cytosolic distribution of a protein probe delivered into live cells. Novel protein probes that can report on the properties of endosomes containing CPP-protein conjugates will also be developed. We anticipate that our results will provide key chemical insights in the critical step of endosomal disruption and lay a firm foundation for the rational design of efficient delivery systems that can achieve cytosolic targeting of protein biosensors with low background of untargeted protein. This will not only enable the microscopy of live cells with externally administered imaging probes but should have an important impact on the entire field of delivery of cell-impermeable macromolecules in general.