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个靶点。已经表明，大量的哺乳动物蛋白质含有特异性结合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，因此重要的是要了解DAG如何发散和非冗余地调节这些蛋白。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标是系统地研究不同的哺乳动物C1结构域及其宿主蛋白如何在体外和哺乳动物细胞中与DAG和含DAG的膜相互作用。这将为我们提供一个重要的线索，DAG分歧调节亚细胞定位和激活的各种DAG受体蛋白的机制，从而建立一个直接的联系之间的复杂的时空动态的DAG和它的生物学效应。该研究计划的长期目标是将从这些研究中学到的原理应用于开发可以特异性调节各种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结构域对可溶性和膜结合DAG和其他脂质的亲和力，并了解其不同配体结合特性的结构基础; 2）建立一个超灵敏，真实的-时间荧光测定法，其允许定量监测和时空分辨细胞DAG信号，对DAG信号传导具有最小抑制作用路径; 3）确定C1结构域和它们的宿主蛋白如何不同地响应于不同水平的细胞DAG，从而确定DAG如何不同地调节各种哺乳动物细胞中多种蛋白的膜靶向和活化。所使用的主要方法包括：1）用于蛋白质-脂质结合的表面等离子体共振、等温滴定量热法和荧光相关光谱分析，以及2）用于实时监测细胞DAG波动和蛋白质-细胞膜结合的各种荧光显微镜技术。

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.