磷脂酶D的结构与分子机制研究

磷脂酶D（PLD）是生物体中普遍存在的一类磷酸二酯酶，能水解多种磷脂产生磷脂酸（PA）等重要信号分子。PLD及其产物PA在动物体生长发育与疾病的发生、植物体逆境响应、激素和防御信号传导等过程中均发挥重要作用。高等生物PLD蛋白由催化结构域和调控结构域组成，其活性受到钙离子和磷脂分子等因素的调控。目前，人们对高等生物PLD的生理功能研究较多，但对于PLD蛋白的底物识别与调控的分子机制尚不清楚。本项目中，我们选择研究广泛的植物体PLD蛋白作为研究对象，利用各种表达体系分离与纯化有活性的目标蛋白，进而解析PLD蛋白及其与底物磷脂、产物磷脂酸复合体的晶体结构，揭示PLD蛋白底物识别和催化的关键分子机制；解析PLD蛋白与活性调控关键因子钙离子、磷脂等小分子抑制剂的三维结构，揭示活性调控的关键位点，阐释PLD活性调控的分子机制。这些研究结果将使我们对PLD蛋白发挥功能的分子机制有一个深入和全新的了解。

Phospholipase D (PLD) is a widely distributed phospholipid hydrolase which catalyzes phospholipids into a number of products, including the important second messengers such as phosphatidic acid (PA). PLD and PA play essential roles in many aspects such as animal development and diseases, plant growth, stress condition, and hormone/defensive signaling. Comprised of a catalytic domain and a regulatory domain, PLD from high organisms hydrolyzes different phospholipids, whose activity is regulated by calcium and specific lipids. Currently, a wealth of research progresses have been made mainly on the biological functions of PLD, but very limited on the three-dimensional structure, and molecular mechanism of substrate recognition, catalysis and regulation. In this project, choosing plant PLD as research target, we will try to obtain the bioactive proteins through recombinant expression and purification, to reveal the molecular mechanism of substrate recognition and catalysis through determination of the PLD-substrate/product complex structures, and to disclose the regulatory mechanism by determining the structure of PLD complexed with regulatory small molecules such as calcium, lipid and inhibitors. A thorough and comprehensive understanding will be made on the molecular mechanism of how PLD functions in high organisms.