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.

Synaptic plasticity plays a crucial role in memory formation by regulating the communication between neurons. Although actin polymerization has been linked to synaptic plasticity and dendritic spine stability, the causal link between actin polymerization and memory encoding has not been identified yet. It is not clear whether actin polymerization and structural changes in dendritic spines are a driver or a consequence of learning and memory. Using an extrinsically disordered form of the protein kinase LIMK1, which rapidly and precisely acts on ADF/cofilin, a direct modifier of actin, we induced long-term enlargement of dendritic spines and enhancement of synaptic transmission in the hippocampus on command. The activation of extrinsically disordered LIMK1 in vivo improved memory encoding and slowed cognitive decline in aged mice exhibiting reduced cofilin phosphorylation. The engineered memory by an extrinsically disordered LIMK1 supports a direct causal link between actin-mediated synaptic transmission and memory.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer’s disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-β precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.