Delivery of protein biosensors across the plasma membrane of live cells

跨活细胞质膜传递蛋白质生物传感器

• 批准号: 8269889
• 负责人: Jean-Philippe Pellois
• 金额: $ 26.5万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269889
• 关键词: Biosensor; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Cytosol; Diagnostic Reagent; Endosomes; Fluorescence; Foundations; Goals; Image; Imaging Techniques; Knowledge; Lead; Learning; Life; Lysosomes; Mammalian Cell; Measures; Mediating; Membrane; Methodology; Microscopy; Modeling; Molecular; Molecular Weight; Morphologic artifacts; Nature; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Population; Problem Solving; Process; Property; Proteins; Reagent; Reporting; Research; Signal Transduction; Structure-Activity Relationship; System; Testing; Time; Viral; Work; abstracting; base; cellular imaging; cytotoxic; cytotoxicity; design; endosome lumen; imaging probe; improved; innovation; insight; macromolecule; nanoparticle; novel; polyhistidine; prevent; protein aminoacid sequence; protein degradation; synthetic protein; tool

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.

项目摘要 我们的长期目标是建立一种方法来有效地将蛋白质传递到活性的细胞质 哺乳动物细胞。当前的输送系统（例如细胞穿透肽（CPP））效率低下，因为 它们促进了其蛋白质货物的广泛内体夹带和降解。这导致 实验性伪像，使蛋白质生物传感器的正确成像不切实际。我们建议解决 通过优化CPP选择性破坏内体膜的能力来实现此问题，以实现 蛋白质从内体更有效地释放到细胞质中并减少降解。我们的具体 目的是：1）确定最佳CPP介导的蛋白质递送所需的条件，2）评估和评估和 优化新型的CPP系统，旨在在酸化的腔内有效破坏膜 内体，3）定义内体pH，CPP浓度和内体释放活性之间的关系 CPP。为了实现这些目标，我们将使用最近开发的成像技术明确 测量输送到活细胞的蛋白质探针的内吞和胞质分布。新蛋白质 可以报​​告包含CPP-蛋白偶联物的内体特性的探针也将是 发达。我们预计我们的结果将在内体的关键步骤中提供关键的化学见解 破坏并为有效交付系统的合理设计奠定了坚定的基础，可以实现 蛋白生物传感器的胞质靶向不靶向的蛋白质背景低。这不仅会启用 具有外部给予成像探针的活细胞的显微镜检查，但应具有重要影响 一般而言，在整个可侵入细胞的大分子的递送领域中。