Developing Biosensors for Signaling Lipids

开发脂质信号生物传感器

• 批准号: 8969216
• 负责人: Sandra M Bajjalieh
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8969216
• 关键词: Affinity; Affinity Chromatography; Algorithms; Animals; Antibodies; Attention; Bacteriophages; Binding; Binding Proteins; Binding Sites; Biosensor; C2 Domain; Calcium; Cell Proliferation; Cell membrane; Cell physiology; Cells; Chimeric Proteins; Controlled Study; Disease; Engineering; Exocytosis; Frequencies; G-Protein-Coupled Receptors; Generations; Goals; Green Fluorescent Proteins; High-Throughput DNA Sequencing; In Situ; In Vitro; Incidence; Inflammation; Lecithin; Libraries; Lipid Bilayers; Lipid Binding; Lipids; Liposomes; Location; Mammalian Cell; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Molecular Machines; Mutagenesis; Mutation; Names; Neurotransmitters; Phage Display; Phosphatidylinositols; Phospholipase A2; Phospholipids; Phosphotransferases; Play; Production; Protein Engineering; Protein Kinase C; Proteins; Receptor Activation; Regulation; Role; Scanning; Second Messenger Systems; Sequence Analysis; Signal Transduction; Signaling Molecule; Site; Solid; Specificity; Surface; Time; Validation; Variant; base; ceramide 1-phosphate; ceramide kinase; deep sequencing; detector; enzyme activity; human PLA2G4A protein; innovation; migration; mutant; nanodisk; novel; novel strategies; protein function; public health relevance; response; scaffold; second messenger; sensor; tool

 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.

 描述（由申请人提供）：脂质是调节细胞功能所有方面的重要信号分子。与蛋白质不同，其位置可以用抗体或标记的融合蛋白的表达原位跟踪，我们原位跟踪信号脂质的能力仍然是原始的，并且构成了理解它们在细胞功能中的作用的主要障碍。这对于信号传导脂质神经酰胺1-磷酸（C1 P）尤其如此，C1 P涉及多种细胞功能，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脂质结合基序的深度扫描诱变，结合深度测序和序列分析算法，以产生与C1 P特异性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跟踪C1 P的基础。此外，我们组装的新型蛋白质工程平台将成为开发其他脂质新传感器的强大新策略。