Molecular Signalling Laboratory, A.I. Virtanen Institute, University of Eastern Finland, Kuopio 70210, Finland.
Neuronal Signalling Laboratory, Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku 20520, Finland.
Nat Commun. 2017 May 12;8:15017. doi: 10.1038/ncomms15017.
Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.
工程化光敏感蛋白调节剂是一项极具多学科挑战性的工作。细胞调节的核心节点 MAPK 的光遗传学调节剂以前尚未被描述。在这里，我们提出了 OptoJNKi，它是一种基于 AsLOV2 光传感器结构域的、使用普遍存在的 FMN 发色团的 JNK 抑制剂。OptoJNKi 的基因转移允许进行光遗传学应用，而蛋白质递送则允许进行光药理学应用。OptoJNKi 的开发为其他光调控抑制剂提供了设计原则。在此基础上，我们生成了 Optop38i，它可以在完整的光照细胞中抑制 p38MAPK。神经元以通过离子通道、膜电位和细胞内钙之间的相互作用来解释时间编码输入而闻名。然而，潜在源自突触活动和可逆转的细胞突起的调节营养输入的 JNK 的时间变化对下游靶标产生的后果是未知的。使用 OptoJNKi，我们发现 c-Jun 转录激活的最大调节可以在出乎意料的缓慢抑制周期发生，这取决于抑制剂的亚细胞位置。这为后生动物 JNK 信号转导回路中的共振提供了证据。
