Genetically Encoded FapR-NLuc as a Biosensor to Determine Malonyl-CoA in Situ at Subcellular Scales
Genetically Encoded FapR-NLuc as a Biosensor to Determine Malonyl-CoA in Situ at Subcellular Scales
复制标题
基因编码的 FapR-NLuc 作为生物传感器，用于在亚细胞尺度上原位测定丙二酰辅酶 A
DOI：
10.1021/acs.bioconjchem.8b00920
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发表时间：
2019-03-01
影响因子：
4.7
通讯作者：
Wei, Taotao
中科院分区：
文献类型：
--
作者：
Du, Yipeng;Hu, Hao;Wei, Taotao
丙二酰辅酶A是脂肪酸代谢的关键代谢中间体之一，也是蛋白质翻译后修饰的关键角色。由于缺乏有效的测量工具，在活细胞中检测丙二酰辅酶A是具有挑战性的。在这里，我们开发了一种基因编码的生物传感器，FAPR-NLuc，通过将丙二酰辅酶A反应细菌转录因子FAPR与工程荧光素酶纳米荧光素酶(NLuc)相结合。FAPR-NLuc对丙二酰辅酶A有特异性的反应，能够在微摩尔水平上快速检测丙二酰辅酶A。更重要的是，它反映了活细胞中丙二酰辅酶A的波动。这种生物传感器定位于亚细胞室，可以在细胞器内原位检测丙二酰辅酶A的变化。因此，FAPR-NLuc有望成为研究丙二酰辅酶A在活细胞中的动力学的一种工具，这将有助于揭示丙二酰辅酶A介导的生物过程的潜在机制。
Malonyl-CoA is one of the key metabolic intermediates in fatty acid metabolism as well as a key player in protein post-translational modifications. Detection of malonyl-CoA in live cells is challenging because of the lack of effective measuring tools. Here we developed a genetically encoded biosensor, FapR-NLuc, by combining a malonyl-CoA responsive bacterial transcriptional factor, FapR, with an engineered luciferase, NanoLuciferase (NLuc). FapR-NLuc specifically responds to malonyl-CoA and enables the rapid detection of malonyl-CoA at the micromolar level. More importantly, it is reflective of the fluctuations of malonyl-CoA in live cells. Upon being targeted to subcellular compartments, this biosensor can detect the changes of malonyl-CoA in situ within organelles. Thus, FapR-NLuc can potentially be used as a tool to study the kinetics of malonyl-CoA in live cells, which will shed light on the underlying mechanisms of malonyl-CoA-mediated biological processes.