Studies of Diacylglycerol-Binding Proteins

描述（由申请人提供）：1,2-二酰基-sn-甘油（DAG）是一种关键的脂质第二信使，介导多种细胞过程，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种靶点。研究表明，大量哺乳动物蛋白含有特异性结合DAG的C1结构域。由于多种DAG结合蛋白存在于许多哺乳动物细胞中，因此了解DAG如何分散和非冗余地调节这些蛋白非常重要。尽管对DAG信号传导的生物学了解很多，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在体外和哺乳动物细胞中与DAG和含DAG膜相互作用的差异。这将为我们揭示DAG差异调控各种DAG受体蛋白亚细胞定位和激活的机制提供重要线索，从而在DAG复杂的时空动态与其生物学效应之间建立直接联系。该研究项目的长期目标是将从这些研究中学到的原理应用于能够特异性调节各种DAG受体的膜靶向和激活的治疗剂的开发。这一阶段的具体目标是：1)定量确定各种C1结构域对可溶性和膜结合的DAG和其他脂质的亲和力，并了解其差异配体结合特性的结构基础；2)建立一种超灵敏的实时荧光分析方法，可以对细胞DAG信号进行定量监测和时空分辨率，同时对DAG信号通路的抑制作用最小；3)确定C1结构域及其宿主蛋白对不同水平细胞DAG的差异反应，从而确定DAG如何在各种哺乳动物细胞中发散调节多种蛋白的膜靶向和激活。主要使用的方法包括：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，用于蛋白质-脂质结合；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-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.