Renna Pietro, Ripoli Cristian, Dagliyan Onur, Pastore Francesco, Rinaudo Marco, Re Agnese, Paciello Fabiola, Grassi Claudio
Department of Neuroscience Università Cattolica del Sacro Cuore Rome Italy.
Fondazione Policlinico Universitario A. Gemelli IRCCS Rome Italy.
Bioeng Transl Med. 2022 Feb 22;7(2):e10292. doi: 10.1002/btm2.10292. eCollection 2022 May.
Engineered proteases are promising tools to address physiological and pathophysiological questions as well as to develop new therapeutic approaches. Here we introduce a new genetically encoded engineered single-chain tobacco etch virus protease, allowing to control proprotein cleavage in different compartments of living mammalian cells. We demonstrated a set of controllable proteolytic effects, including cytosolic protein cleavage, inducible gene expression, and maturation of brain-derived neurotrophic factor (BDNF) in the secretory pathway thus showing the versatility of this technique. Of note, the secretory pathway exhibits different characteristics from the cytosol and it is difficult to target because inaccessible to some small molecules. We were able to induce ligand-mediated BDNF maturation and monitor its effects on dendritic spines in hippocampal pyramidal cells and in the mouse brain. This strategy paves the way to dissect proteolytic cleavage product signaling in various processes as well as for future therapeutic applications.
工程化蛋白酶是解决生理和病理生理问题以及开发新治疗方法的有前景的工具。在此，我们介绍一种新的基因编码工程化单链烟草蚀纹病毒蛋白酶，它能够控制活的哺乳动物细胞不同区室中的前体蛋白切割。我们展示了一系列可控的蛋白水解效应，包括胞质蛋白切割、诱导型基因表达以及分泌途径中脑源性神经营养因子（BDNF）的成熟，从而显示了该技术的多功能性。值得注意的是，分泌途径具有与胞质溶胶不同的特征，并且由于一些小分子无法进入而难以靶向。我们能够诱导配体介导的BDNF成熟，并监测其对海马锥体细胞和小鼠脑中树突棘的影响。这一策略为剖析各种过程中蛋白水解切割产物信号传导以及未来的治疗应用铺平了道路。
