Developing Biosensors for Signaling Lipids

 描述(申请人提供)：脂类是调节细胞各方面功能的重要信号分子。与蛋白质不同，我们可以用抗体或标记融合蛋白的表达来原位追踪其位置，我们在原位追踪信号脂类的能力仍然是原始的，并构成了解它们在细胞功能中作用的主要障碍。尤其是信号转导的脂质神经酰胺1-磷酸(C1P)，它参与了多种细胞功能，包括递质胞吐、炎症、细胞增殖、蛋白质功能调节和细胞骨架动力学。为了克服这一技术挑战，我们将使用C2脂结合基序的深度扫描突变，结合深度测序和序列分析算法，产生与C1P特异、高亲和力结合的蛋白质。由此产生的蛋白质将作为荧光生物传感器原位追踪C1P的基础。此外，我们组装的新型蛋白质工程平台将成为开发用于其他脂质的新传感器的有力新策略。

 DESCRIPTION (provided by applicant): Lipids are essential signaling molecules that regulate all aspects of cellular function. Unlike proteins, whose location can be tracked in situ with antibodies or expression of tagged fusion proteins, our ability to track signaling lipids in situ i still primitive and constitutes a major impediment to understanding their role in cell function. Ths is especially true for the signaling lipid ceramide 1- phosphate (C1P), which has been implicated in multiple cellular functions including transmitter exocytosis, inflammation, cell proliferation, protein function regulation, and cytoskeletal dynamics. To overcome this technological challenge we will use deep scanning mutagenesis of the C2 lipid-binding motif, combined with deep sequencing and sequence analysis algorithms, to generate proteins with specific, high-affinity binding to C1P. The resulting proteins will serve as the basis for fluorescent biosensors to track C1P in situ. In addition, the novel protein engineering platform we assemble will be a powerful new strategy for developing new sensors for other lipids.