Studies of Diacylglycerol-Binding Proteins

二酰甘油结合蛋白的研究

• 批准号: 7149837
• 负责人: WONHWA CHO
• 金额: $ 29.1万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149837
• 关键词: binding proteins; biological signal transduction; computer simulation; diacylglycerols; gastrin releasing peptide; gene expression; gene mutation; glycerol kinase; guanine nucleotide binding protein; intermolecular interaction; lipid metabolism; lipid solubility; membrane lipids; phorbols; protein kinase C; protein structure function

DESCRIPTION (provided by applicant): 1,2-diacyl-sn-glycerol (DAG) is a key lipid second messenger that mediates a wide variety of cellular processes, including cell proliferation and malignant transformation. Therefore, DAG signaling pathways offer multiple targets for anticancer therapy. It has been shown that a significant number of mammalian proteins contain the C1 domain that specifically binds DAG. Because multiple DAG-binding proteins are present in many mammalian cells, it is important to understand how divergently and non-redundantly DAG regulates these proteins. Although much is known about the biology of DAG signaling, less is known about the quantitative aspects of ligand and membrane binding of DAG-receptor proteins and the spatiotemporal dynamics of the cellular DAG. The primary objective of this research projects is to systematically study how differentially various mammalian C1 domains and their host proteins interact with DAG and DAG-containing membranes both in vitro and in mammalian cells. This will provide us with an important clue to the mechanisms by which DAG divergently regulates the subcellular localization and activation of various DAG receptor proteins, and will thereby establish a direct link between the complex spatiotemporal dynamics of DAG and its biological effects. A long-term objective of this research program is to apply the principles learned from these studies to the development of therapeutic agents that can specifically modulate the membrane targeting and activation of various DAG receptors. Specific aims for this period are: 1) To quantitatively determine affinities of various C1 domains for soluble and membrane-incorporated DAG and other lipids and to understand the structural basis of their differential ligand binding properties; 2) To establish an ultra-sensitive, real-time fluorometric assay that allows for quantitative monitoring and spatiotemporal resolution of cellular DAG signals with a minimal inhibitory effect on DAG signaling pathways; 3) to determine how differentially C1 domains and their host proteins respond to different levels of cellular DAG and thereby determine how DAG divergently regulates the membrane targeting and activation of multiple proteins in various mammalian cells. The principal methodologies to be used include: 1) surface plasmon resonance, isothermal titration calorimetry, and fluorescence correlation spectroscopy analyses for protein-lipid binding and 2) various fluorescence microscopy techniques for real-time monitoring of cellular DAG fluctuation and protein-cell membrane binding.

描述（由申请人提供）：1,2-二酰基-SN-甘油（DAG）是一个关键的脂质第二信使，可介导各种细胞过程，包括细胞增殖和恶性转化。因此，DAG信号通路为抗癌治疗提供了多个目标。已经表明，大量的哺乳动物蛋白包含专门结合DAG的C1结构域。由于许多哺乳动物细胞中存在多种DAG结合蛋白，因此重要的是要了解如何发散和非差异DAG调节这些蛋白质。尽管对DAG信号的生物学知之甚少，但对DAG受体蛋白的配体和膜结合的定量方面以及细胞DAG的时空动力学的了解却鲜为人知。该研究项目的主要目的是系统地研究各种哺乳动物C1结构域及其宿主蛋白如何在体外和哺乳动物细胞中与含DAG和含DAG的膜相互作用。这将为我们提供一个重要的线索，即DAG调节各种DAG受体蛋白的亚细胞定位和激活的机制，从而在复杂的DAG的复杂时空动力学及其生物学效应之间建立直接联系。该研究计划的一个长期目标是将从这些研究学到的原则应用于可以特异性调节各种DAG受体的膜靶向和激活的治疗剂的开发。这一时期的具体目的是：1）定量确定各种C1结构域的亲和力，用于可溶性和膜上的DAG和其他脂质，并了解其差异配体结合特性的结构基础； 2）建立一个超敏感的实时荧光测定法，该测定法可以对细胞DAG信号进行定量监测和时空分辨率，对DAG信号通路的抑制作用最少； 3）确定差异性C1结构域及其宿主蛋白如何对不同水平的细胞DAG做出反应，从而确定DAG如何分散调节各种哺乳动物细胞中多种蛋白的膜靶向和激活。要使用的主要方法包括：1）表面等离子体共振，等温滴定量热法和荧光相关光谱分析蛋白 - 磷结合和2）各种荧光显微镜技术，用于实时监测细胞DAG波动和蛋白质 - 膜膜膜膜结合的实时监测。