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. ? ? ?
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