Improvement of Phycocyanobilin Synthesis for Genetically Encoded Phytochrome-Based Optogenetics
Improvement of Phycocyanobilin Synthesis for Genetically Encoded Phytochrome-Based Optogenetics
复制标题
用于基因编码光敏色素光遗传学的藻蓝蛋白合成的改进
DOI：
10.1021/acschembio.0c00477
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发表时间：
2020
影响因子：
4
通讯作者：
Aoki Kazuhiro
中科院分区：
文献类型：
--
作者：
Uda Youichi;Miura Haruko;Goto Yuhei;Yamamoto Kei;Mii Yusuke;Kondo Yohei;Takada Shinji;Aoki Kazuhiro
光遗传学是一种使用光响应蛋白的强大技术，光诱导二聚化（LID）系统是一种光遗传学工具，可以操纵细胞内信号传导途径。光敏色素B（PhyB）-光敏色素相互作用因子（PIF）是一种红色/远红色响应的LID系统，具有在第二时间尺度上控制光的缔合和解离的独特性质，但PhyB需要线性四吡咯发色团如藻蓝胆素（PCB），而此类发色团仅存在于高等植物和蓝藻中。在这里，我们报告说，我们进一步改进了我们以前开发的PCB合成系统（SynPCB），并成功地建立了一个稳定的细胞系含有遗传编码的PhyB-PIF LID系统。首先，四个负责PCB合成的基因，即PcyA，HO 1，Fd和Fnr，被替换为来自嗜热蓝藻的对应基因。其次，Fnr被截短，随后与Fd融合以产生嵌合蛋白tFnr-Fd。第三，将这些基因与P2 A肽cDNA串联用于多顺反子表达，导致PCB合成与先前版本相比增加约4倍。最后，我们将PhyB，PIF和SynPCB系统整合到药物诱导慢病毒和转座子载体中，这使得我们能够通过强力霉素处理诱导PCB合成和PhyB-PIF LID系统。这些工具提供了一个新的机会来促进我们对细胞内信号传导和细胞功能之间因果关系的理解。
Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)–phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely,PcyA,HO1,Fd, andFnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.