A Bioengineering Strategy to Control ADAM10 Activity in Living Cells

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease\r\nubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in\r\nmultiple physiological processes. The dysregulation of ADAM10 expression and function has been\r\nimplicated in pathological conditions, including Alzheimer’s disease (AD). Although it has been\r\nsuggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its\r\nactivation, other potential mechanisms for the ADAM10 proteolytic function and activation remain\r\nunclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain,\r\nwhich regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and\r\ntemporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated\r\ncleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy\r\nto control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the\r\nintervention of small endogenous molecule signaling pathways. We generated a series of engineered\r\nADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering\r\nADAM10 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\r\ncleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated\r\nADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence\r\nof TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy\r\nfor controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of\r\nADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity\r\nand the non-amyloidogenic cleavage of amyloid-β precursor protein (APP), thereby increasing the\r\nproduction of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the\r\npotential to be exploited as a next-generation gene therapy for AD