Protein Translocation Using Chimeric Toxins

有了这个奖项，化学部的生命过程化学项目正在资助韦恩州立大学的安德鲁·菲格博士研究一种新的方法，使用嵌合蛋白质，可以自发地运输自己穿过真核细胞的细胞膜。嵌合蛋白将使用基于艰难梭菌毒素的支架构建。这些毒素通过转运葡糖基转移酶杀死宿主细胞，葡糖基转移酶通过修饰细胞G蛋白启动细胞凋亡。Feig团队致力于去除天然催化结构域，并用设计的货物取代它。预计这将允许递送代谢活性蛋白质来代替天然货物结构域。嵌合体将被优化以改善其递送特性，并测试该技术可以通过直接向细胞递送蛋白质来调节细胞功能的极限。此外，正在探索用识别特定受体的片段取代通常与细胞表面结合的毒素结构域的可能性。 目标是有效地将货物递送仅针对那些表达指定伴侣的细胞。这将实现蛋白质递送的圣杯，因为它将提供可调的细胞选择性并将无标签蛋白质直接递送到胞质区室。多年来，科学家们一直在操纵细胞来研究单个蛋白质对细胞功能的贡献。然而，这种方法的一个主要限制是，它仅限于细胞中可用的那些构建模块，因为蛋白质仍然由核糖体翻译。这项工作将进一步开发最近由Feig实验室提出的技术，该技术允许适当设计的蛋白质穿过细胞膜运输自己，在细胞溶质中释放活性蛋白，而没有任何残留的标签。这种技术有许多潜在的用途，包括蛋白质治疗剂的靶向递送、可以在细胞内容易地跟踪以研究其命运的修饰蛋白质的递送以及细胞途径的操纵。该项目将与主要研究者参与的教师发展工作相结合，培训化学和生物化学领域的新教师，以便在课堂上使用循证教学方法，并更好地评估学生的学习质量实时。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Andrew Feig from Wayne State University to study a new approach to using chimeric proteins that can spontaneously transport themselves across the cellular membrane of eukaryotic cells. The chimeric proteins will be built using a scaffold based on toxins from Clostridium difficile. These toxins kill host cells by transporting a glucosyltransferase that initiates apoptosis by modifying cellular G-proteins. The Feig team works to remove the natural catalytic domain and replace it with designed cargos. This is expected to allow for the delivery of metabolically active proteins in lieu of the natural cargo domain. Chimeras are to be optimized to improve their delivery properties and test the limits to which this technology can be pushed to regulate cellular functions by direct protein delivery to cells. In addition, the possibility to replace the domain of the toxin that normally binds the cell surface with fragments that recognize specific receptors is being explored. The goal is to effectively target cargo delivery to only those cells expressing the designated partner. This would achieve a holy grail of protein delivery since it would provide tunable cell selectivity and deliver tagless proteins directly to the cytosolic compartment. Scientists have, for many years, manipulated cells to study the way in which individual proteins contribute to cellular functions. However, a major limitation of this approach is that one is limited to those building blocks available in the cell since the proteins are still translated by the ribosome. This work will further develop technology that was recently put forth by the Feig Lab that allows properly designed proteins to transport themselves across the cell membrane releasing an active protein in the cytosol without any residual tags. There are many potential uses for such technology including the targeted delivery of protein therapeutics, the delivery of modified proteins that can be easily tracked within the cell to study their fate and the manipulation of cellular pathways. The project will be integrated with faculty development work that the principal investigator is involved with that trains new faculty in the fields of Chemistry and Biochemistry to use evidence-based teaching methods in the classroom and to better assess the quality of student learning in real-time.