Trifunctional lipid probes for comprehensive studies of single lipid species in living cells
Trifunctional lipid probes for comprehensive studies of single lipid species in living cells
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DOI：
10.1073/pnas.1611096114
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发表时间：
2017-02-14
影响因子：
11.1
通讯作者：
Schultz, Carsten
中科院分区：
文献类型：
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作者：
Hoeglinger, Doris;Nadler, Andre;Schultz, Carsten
脂质介导的信号传导事件调节许多细胞过程。调查的复杂的基本机制是困难的，因为几个不同的方法需要在不同的条件下使用。在这里，我们介绍了多功能的脂质衍生物，研究脂质代谢，脂质-蛋白质相互作用，和细胞内脂质定位与一个单一的工具，每个目标脂质。探针配备有两个光反应性基团，以允许以顺序方式光致电离（释放）和光交联，以及用于后续功能化的点击手柄。我们证明了信号脂质鞘氨醇和甘油二酯的设计的多功能性;这两个物种的探针的释放分别触发了钙信号和细胞内蛋白质易位事件。我们进行了蛋白质组学筛选，以绘制两种脂质的脂质相互作用蛋白质组。最后，我们可视化鞘氨醇转运缺陷患者来源的尼曼-匹克病C型成纤维细胞的荧光以及相关的光学和电子显微镜，指向这些工具的诊断潜力。我们设想，这种类型的探针将成为重要的分析和最终了解脂质信号事件的全面方式。
Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid-protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo-cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann-Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.