Fluorogenic Assays for Measuring the Activity of Ceramidase and Sphingomyelinase

描述（由申请人提供）：鞘脂是一类脂类，作为细胞膜的结构成分具有重要作用，同时也是参与许多细胞过程的关键信号分子，如凋亡，细胞存活，分化，运动和生长。各种鞘脂的水平通过代谢酶保持平衡，这些酶的异常水平会导致疾病，如癌症、糖尿病、动脉硬化和自身免疫性疾病。在此应用中，将开发两种重要的鞘脂代谢酶，神经酰胺酶和鞘磷脂酶的荧光检测方法。荧光去猝灭试验利用内部猝灭的荧光底物，当被酶水解时，由于猝灭剂不再靠近荧光而发出荧光。神经酰胺和鞘磷脂的标记类似物将被合成，用它们各自的酶作为底物进行评价，并优化检测条件。这些检测方法可用于进一步的基础研究，并适用于治疗性化合物的高通量筛选。

DESCRIPTION (provided by applicant): Sphingolipids are a class of lipids that have important roles as structural components of cell membranes yet are also key signaling molecules involved in numerous cellular processes such as apoptosis, cell survival, differentiation, motility, and growth. The levels of the various sphingolipids are kept in balance by metabolizing enzymes and disruptions by abnormal levels of these enzymes are known to occur in diseases such as cancer, diabetes, arthersclerosis, and autoimmune disorders. In this application, fluorogenic assays for two important sphingolipid metabolizing enzymes, ceramidase and sphingomyelinase, will be developed. The fluorescence dequenching assays utilize internally quenched fluorescent substrates which, when hydrolyzed by the enzymes, fluoresce due to the quencher no longer being in close proximity to the fluor. Labeled analogs of ceramide and sphingomyelin will be synthesized, evaluated as substrates with their respective enzymes, and the assay conditions will be optimized. These assays can be employed to further basic research and be adapted to high throughput screening for therapeutic compounds. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to develop assays for two important lipid metabolizing enzymes: ceramidase and sphingomyelinase. These assays will help to further basic research in this growing field and potentially be used for developing new drugs for diseases such as cancer, diabetes, and Alzheimer's disease.