Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling
Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling
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DOI：
10.1073/pnas.1707190114
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发表时间：
2017-10
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影响因子：
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通讯作者：
Y. Uda;Yuhei Goto;S. Oda;T. Kohchi;M. Matsuda;K. Aoki
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作者：
Y. Uda;Yuhei Goto;S. Oda;T. Kohchi;M. Matsuda;K. Aoki
近年来，细胞信号的光调控引起了人们的广泛关注。大多数光遗传学系统利用蓝光，荧光指示剂如基于FRET的生物传感器也是如此。光敏色素B（PhyB）-光敏色素相互作用因子（PIF）是一个红光/远红光可转换的异源二聚体系统。然而，PhyB需要phytochromobilin或phycocyanobilin（PCB）作为发色团，必须从外部添加到哺乳动物细胞。为了克服这个困难，我们开发了一个系统，在哺乳动物细胞中的PCB的有效合成。最终，我们发现PCB是通过引入所有的PcyA，HO 1，Fd和Fnr基因合成的。胆绿素还原酶A的敲除或敲低进一步增强PCB合成。这种基因编码的PCB合成系统使我们能够在没有外源PCB添加的情况下通过红光/远红光操纵细胞信号传导。光遗传学是一种在空间和时间上精确操纵细胞信号的强大工具。例如，蛋白质活性可以通过几种光诱导二聚化（LID）系统来调节。其中光敏色素B（PhyB）-光敏色素相互作用因子（PIF）系统是目前唯一可用的红光和远红光控制的LID系统。然而，PhyB-PIF系统需要藻蓝胆素（PCB）或phytochromobilin作为发色团，必须人工添加到哺乳动物细胞中。在这里，我们报告了一个表达载体，共表达HO 1和PcyA与铁氧还蛋白和铁氧还蛋白NADP+还原酶的PCB在哺乳动物细胞的线粒体中的有效合成。甚至更高的细胞内PCB浓度是通过消耗胆绿素还原酶A来实现的，胆绿素还原酶A降解PCB。PCB合成和PhyB-PIF系统允许我们在没有任何外部发色团供应的情况下光遗传学地调节细胞内信号传导。因此，我们提供了一种实用的方法来开发一个完全遗传编码的PhyB-PIF系统，这为其应用于活体动物铺平了道路。
Significance Optical control of cell signaling has attracted much attention in recent years. Most optogenetic systems exploit blue light, as do fluorescent indicators such as FRET-based biosensors. Phytochrome B (PhyB)–phytochrome-interacting factor (PIF) is a red/far-red light-switchable heterodimerization system. However, PhyB requires phytochromobilin or phycocyanobilin (PCB) as a chromophore, which must be externally added to mammalian cells. To overcome this difficulty, we developed a system for an efficient synthesis of PCB in mammalian cells. Eventually, we found that PCB was synthesized by introducing all PcyA, HO1, Fd, and Fnr genes. The knockout or knockdown of biliverdin reductase A further enhanced PCB synthesis. This genetically encoded PCB synthesis system allowed us to manipulate cell signaling by red/far-red light without exogenous PCB addition. Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)–phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB–PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB–PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB–PIF system, which paves the way for its application to a living animal.