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，有效地将蛋白质递送到活细胞的胞质溶胶中， 哺乳动物细胞目前的递送系统如细胞穿透肽（CPP）是低效的，因为 它们促进广泛的内体截留和它们的蛋白质货物的降解。这导致 使蛋白质生物传感器的适当成像不切实际的实验伪像。我们建议解决 通过优化CPP选择性破坏内体膜的能力，以实现 更有效地将蛋白质从内体释放到胞质溶胶中并减少降解。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 优化新型CPP系统，该系统设计用于在管腔酸化时有效地破坏膜， 内体，3）定义内体pH、CPP浓度和内体释放活性之间的关系 的CPP。为了实现这些目标，我们将使用最近开发的成像技术， 测量递送到活细胞中的蛋白质探针的内吞和胞质溶质分布。新蛋白质 还将使用能够报告含有CPP-蛋白缀合物的内体性质的探针， 开发我们预计，我们的研究结果将提供关键的化学见解的关键步骤，内体 中断，并为合理设计高效的交付系统奠定坚实的基础， 蛋白质生物传感器的胞质靶向，具有低背景的非靶向蛋白质。这不仅可以使 活细胞的显微镜与外部管理的成像探针，但应该有重要的影响， 在整个领域的传递细胞不可渗透的大分子一般。