Department of Chemistry and Chemical Biology and Weill Institute for Cell and Molecular Biology, Cornell University, 464 Weill Hall, Ithaca, NY, 14853, USA.
Angew Chem Int Ed Engl. 2016 Oct 10;55(42):13155-13158. doi: 10.1002/anie.201607443.
Phosphatidic acid (PA) is a potent lipid secondary messenger, the synthesis of which is tightly regulated in both space and time. Established tools for detecting PA involve ex vivo analysis and do not provide information on the subcellular locations where this lipid is synthesized. Here, a chemoenzymatic strategy for imaging sites of cellular PA synthesis by phospholipase D (PLD) enzymes is reported. PLDs were found to be able to catalyze phospholipid head-group exchange with alkynols to generate alkyne-labeled PA analogues within cells. Subsequent fluorophore tagging through Cu-catalyzed azide-alkyne cycloaddition enabled both visualization by fluorescence microscopy and quantification by HPLC. Our studies revealed several intracellular sites of PLD-mediated PA synthesis. We envision applications of this approach to dissect PA-dependent signaling pathways, image PLD activity in disease, and remodel intracellular membranes with new functionality.
磷酸脂酸（PA）是一种有效的脂类二级信使，其合成在空间和时间上受到严格调控。已有的检测 PA 的工具涉及体外分析，无法提供这种脂质在何处合成的亚细胞位置信息。本研究报告了一种通过磷脂酶 D（PLD）酶对细胞内 PA 合成部位进行化学酶法成像的策略。发现 PLD 能够催化与炔醇的磷脂头部基团交换，在细胞内生成炔基标记的 PA 类似物。随后通过铜催化的叠氮-炔环加成反应进行荧光标记，可通过荧光显微镜进行可视化观察，通过 HPLC 进行定量分析。我们的研究揭示了几个 PLD 介导的 PA 合成的细胞内部位。我们设想这种方法可用于剖析 PA 依赖的信号通路、在疾病中成像 PLD 活性，以及用新功能重塑细胞内膜。
