Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):11962-11967. doi: 10.1073/pnas.1707190114. Epub 2017 Oct 24.
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.
光遗传学是一种精确操纵细胞信号时空的强大工具。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，植物色素 B（PhyB）-植物色素相互作用因子（PIF）系统是唯一可用的受红光和远红光控制的 LID 系统。然而，PhyB-PIF 系统需要藻胆胆素（PCB）或植物胆素作为生色团，这必须人工添加到哺乳动物细胞中。在这里，我们报告了一种表达载体，该载体共表达 HO1 和 PcyA 与铁氧还蛋白和铁氧还蛋白-NADP+还原酶，以在哺乳动物细胞的线粒体中高效合成 PCB。通过耗尽降解 PCB 的胆红素还原酶 A，实现了更高的细胞内 PCB 浓度。PCB 的合成和 PhyB-PIF 系统使我们能够进行光遗传学调节细胞内信号，而无需任何外部生色团供应。因此，我们为开发完全基因编码的 PhyB-PIF 系统提供了一种实用方法，为其在活体动物中的应用铺平了道路。
