鞘脂代谢途径关键酶ACD5的结构解析及其功能调控的分子机制

鞘脂不仅是生物膜的主要结构成分，也是重要的生物活性分子。神经酰胺是鞘脂代谢途径的中心分子，其积累水平直接影响细胞的生死和对生物和非生物胁迫的应答能力。神经酰胺激酶ACD5是植物中调控神经酰胺水平的关键酶，本申请是在我们前期大量研究的基础上，深入研究ACD5的蛋白结构、活性位点及其功能调控的分子机制。主要探讨以下内容：1）采用冷冻电镜技术解析ACD5的蛋白结构特点； 2）利用高灵敏和高分辨率液质联用技术，建立微量（或低量）鞘脂的检测方法和平台，研究ACD5的体内酶活及关键酶活位点；3）利用遗传、生化等手段，研究ACD5的互作蛋白及与互作蛋白的调控关系。本申请将有助于在蛋白结构层面上揭示和理解神经酰胺激酶的功能及调控机制，为未来该蛋白在植物逆境应答中的有效利用提供理论依据。

Sphingolipids are major structural components of plasma membrane and endomembrane system, and also important bioactive molecules. Ceramide is the central molecule of sphingolipid metabolism pathway, and its accumulation level directly affects plant programmed cell death and biotic and abiotic responses. Ceramide kinase ACD5, as a key enzyme in sphingolipid metabolism, regulates ceramide level in plant cells. This proposal is based on our previous studies to further explore the ACD5 protein structures, active sites of the enzyme and the molecular mechanism of its function. The contents of the application will mainly cover three parts as follows: 1) By using Cryo-EM, we will analyze the structural basis of ACD5 protein; 2) Using high sensitive and high resolution of LC/MS, we will establish a micro sphingolipid detection platform, and study the activity of ACD5 in vivo and active sites of the enzyme; 3) By combination of genetics and biochemical tools, we will screen and study the ACD5 interaction proteins and its regulation. This proposal will help us to reveal and comprehensive understand the function and regulatory mechanism of ceramide kinase, and provide a theoretical basis for its effective utilization in plant stress responses in the future.