Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
Interfaculty Doctoral Study of Biomedicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
ACS Synth Biol. 2022 Aug 19;11(8):2756-2765. doi: 10.1021/acssynbio.2c00151. Epub 2022 Jul 8.
Highly regulated intracellular calcium entry affects numerous cellular physiological events. External regulation of intracellular calcium signaling presents a great opportunity for the artificial regulation of cellular activity. Calcium entry can be mediated by STIM proteins interacting with Orai calcium channels; therefore, the STIM1-Orai1 pair has become a tool for artificially modulating calcium entry. We report on an innovative genetically engineered protease-activated Orai activator called PACE. CAD self-dimerization and activation were inhibited with a coiled-coil forming peptide pair linked to CAD via a protease cleavage site. PACE generated sustained calcium entry after its activation with a reconstituted split protease. We also generated PACE, whose transcriptional activation of NFAT was triggered by PPV or TEV protease. Using PACE, we successfully activated the native NFAT signaling pathway and the production of cytokines in a T-cell line. PACE represents a useful tool for generating sustained calcium entry to initiate calcium-dependent protein translation. PACE provides a promising template for the construction of links between various protease activation pathways and calcium signaling.
高度调控的细胞内钙离子内流影响众多细胞生理事件。细胞内钙信号的外部调节为细胞活动的人工调节提供了绝佳机会。钙离子内流可以通过与 Orai 钙通道相互作用的 STIM 蛋白来介导；因此，STIM1-Orai1 对成为人工调节钙离子内流的工具。我们报告了一种名为 PACE 的创新基因工程蛋白酶激活的 Orai 激活剂。CAD 自身二聚化和激活被通过蛋白酶切割位点连接到 CAD 的卷曲螺旋形成肽对抑制。PACE 在经过重组的分裂蛋白酶激活后产生持续的钙离子内流。我们还生成了 PACE，其 NFAT 的转录激活由 PPV 或 TEV 蛋白酶触发。使用 PACE，我们成功地激活了 T 细胞系中的天然 NFAT 信号通路和细胞因子的产生。PACE 是一种有用的工具，可用于产生持续的钙离子内流以启动钙依赖性蛋白翻译。PACE 为构建各种蛋白酶激活途径与钙信号之间的联系提供了有前途的模板。
