Zhao Jingyi, Liu Yanjun, Lin Feng, Wang Weixia, Yang Shaojun, Ge Yun, Chen Peng R
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
ACS Cent Sci. 2019 Jan 23;5(1):145-152. doi: 10.1021/acscentsci.8b00751. Epub 2018 Dec 27.
The complicated and entangled cell signaling network is dynamically regulated by a wide array of enzymes such as kinases. It remains desirable but challenging to specifically modulate individual, endogenous kinases within a cell, particularly in a spatial-temporally controlled fashion. Current strategies toward regulating the intracellular functions of a kinase of interest either lack specificity or require genetic engineering that may perturb its physiological activity. Herein, we harnessed a bacterial effector OspF for optical and chemical modulation of the endogenous mitogen-activated protein kinase (MAPK) cascade in living cells and mice. The phospho-lyase OspF provided high specificity and spatial resolution toward the desired kinase such as the extracellular signal-regulated kinase (ERK), while the genetically encoded bioorthogonal decaging strategy enabled its temporal activation in living systems. The photocaged OspF (OspF*) was applied to dissect the subcellular signaling roles of ERK in nucleus as opposed to cytoplasm, while the chemically caged OspF (OspF) was introduced into living mice to modulate ERK-mediated gene expression. Finally, our spatially and chemically controlled OspF was further used to precisely tune immune responses in T cells. Together, our bioorthogonal engineering strategy on bacterial effectors offers a general tool to modulate cell signaling with high specificity and spatial-temporal resolution.
复杂且相互缠绕的细胞信号网络受到多种酶（如激酶）的动态调控。在细胞内特异性地调节单个内源性激酶，尤其是以时空可控的方式进行调节，仍然是人们所期望但具有挑战性的。目前用于调节目标激酶细胞内功能的策略要么缺乏特异性，要么需要可能干扰其生理活性的基因工程。在此，我们利用细菌效应蛋白OspF对活细胞和小鼠体内的内源性丝裂原活化蛋白激酶（MAPK）级联反应进行光学和化学调节。磷酸裂解酶OspF对所需激酶（如细胞外信号调节激酶（ERK））具有高特异性和空间分辨率，而基因编码的生物正交脱笼策略使其能够在生物系统中进行时间激活。光笼化的OspF（OspF*）被用于剖析ERK在细胞核与细胞质中不同的亚细胞信号传导作用，而化学笼化的OspF（OspF）被引入活小鼠体内以调节ERK介导的基因表达。最后，我们的空间和化学可控的OspF被进一步用于精确调节T细胞中的免疫反应。总之，我们对细菌效应蛋白的生物正交工程策略提供了一种以高特异性和时空分辨率调节细胞信号传导的通用工具。
