A Bioengineering Strategy to Control ADAM10 Activity in Living Cells
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 patholog...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/2/917 |
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| author | Francesco Pastore Martina Battistoni Raimondo Sollazzo Pietro Renna Fabiola Paciello Domenica Donatella Li Puma Eugenio Barone Onur Dagliyan Cristian Ripoli Claudio Grassi |
| author_facet | Francesco Pastore Martina Battistoni Raimondo Sollazzo Pietro Renna Fabiola Paciello Domenica Donatella Li Puma Eugenio Barone Onur Dagliyan Cristian Ripoli Claudio Grassi |
| author_sort | Francesco Pastore |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T12:27:32Z |
| format | Article |
| id | doaj.art-352a4edfaf5840c8a0adff688c7bc10e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T12:27:32Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-352a4edfaf5840c8a0adff688c7bc10e2023-11-30T22:32:28ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-01-0124291710.3390/ijms24020917A Bioengineering Strategy to Control ADAM10 Activity in Living CellsFrancesco Pastore0Martina Battistoni1Raimondo Sollazzo2Pietro Renna3Fabiola Paciello4Domenica Donatella Li Puma5Eugenio Barone6Onur Dagliyan7Cristian Ripoli8Claudio Grassi9Department of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyDepartment of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyDepartment of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyDepartment of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyDepartment of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyDepartment of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyDepartment of Biochemical Sciences “A. Rossi-Fanelli”, Sapienza University of Rome, 00185 Rome, ItalyDivision of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 65 Stockholm, SwedenDepartment of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyDepartment of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Rome, ItalyA 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.https://www.mdpi.com/1422-0067/24/2/917prodomaincytosolic domainTEVengineered proteinAPPAlzheimer |
| spellingShingle | Francesco Pastore Martina Battistoni Raimondo Sollazzo Pietro Renna Fabiola Paciello Domenica Donatella Li Puma Eugenio Barone Onur Dagliyan Cristian Ripoli Claudio Grassi A Bioengineering Strategy to Control ADAM10 Activity in Living Cells International Journal of Molecular Sciences prodomain cytosolic domain TEV engineered protein APP Alzheimer |
| title | A Bioengineering Strategy to Control ADAM10 Activity in Living Cells |
| title_full | A Bioengineering Strategy to Control ADAM10 Activity in Living Cells |
| title_fullStr | A Bioengineering Strategy to Control ADAM10 Activity in Living Cells |
| title_full_unstemmed | A Bioengineering Strategy to Control ADAM10 Activity in Living Cells |
| title_short | A Bioengineering Strategy to Control ADAM10 Activity in Living Cells |
| title_sort | bioengineering strategy to control adam10 activity in living cells |
| topic | prodomain cytosolic domain TEV engineered protein APP Alzheimer |
| url | https://www.mdpi.com/1422-0067/24/2/917 |
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