Studies of Diacylglycerol-Binding Proteins

描述(申请人提供)：1，2-二酰基-sn-甘油(DAG)是一种重要的脂类第二信使，调节多种细胞过程，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靶点。已有研究表明，许多哺乳动物蛋白质含有与DAG特异结合的C1域。由于许多哺乳动物细胞中存在多种DAG结合蛋白，因此了解DAG如何以不同和非冗余的方式调节这些蛋白是很重要的。虽然对DAG信号的生物学了解很多，但对DAG受体蛋白的配体和膜结合的定量方面以及细胞DAG的时空动力学知之甚少。本研究的主要目的是在体外和哺乳动物细胞中系统地研究不同的哺乳动物C1结构域及其宿主蛋白如何与DAG和含有DAG的膜相互作用。这将为我们提供一条重要的线索，揭示DAG不同地调节各种DAG受体蛋白的亚细胞定位和激活的机制，从而在DAG复杂的时空动力学及其生物学效应之间建立直接的联系。这项研究计划的一个长期目标是将从这些研究中学到的原理应用于开发能够特异性调节各种DAG受体的膜靶向和激活的治疗剂。这一时期的具体目标是：1)定量测定各种C1结构域与可溶性和膜结合DAG及其他脂类的亲和力，并了解其差异配体结合特性的结构基础；2)建立一种超灵敏、实时的荧光分析方法，能够对细胞DAG信号进行定量监测和时空分辨率分析，同时将对DAG信号通路的抑制作用降至最低；3)确定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如何在不同的哺乳动物细胞中对多种蛋白质的膜靶向和激活进行不同的调节。主要使用的方法包括：1)表面等离子体共振法、等温滴定量热法和荧光相关光谱分析蛋白质-脂结合；2)各种荧光显微镜技术实时监测细胞DAG波动和蛋白质-细胞膜结合。

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.