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Here we introduce ProNotch, a receptor architecture that harnesses protease-gated derepression of a mutant Notch1 negative regulatory region (NRR) to drive user-defined gene expression. ProNotch tethers destabilized NRR mutants to inhibitory anti-NRR single-chain variable fragments (scFvs) via protease-cleavable linkers. NRR engagement by high-affinity scFvs simultaneously rescues surface trafficking of mutant receptors and suppresses basal signaling until linker cleavage releases the inhibitory scFv module, permitting the destabilized NRR to initiate ligand-independent signaling. Linker substitution reprogrammed protease specificity across diverse enzymes, and tandem substrate repeats enhanced sensitivity without increasing basal activity. Single-chain receptor designs enabled OR and AND logic gates, allowing integration of multi-protease inputs into a single transcriptional output. ProNotch detected endogenous MMP-14 activity from cancer cell lines in\n                  <jats:italic>cis<\/jats:italic>\n                  and in\n                  <jats:italic>trans<\/jats:italic>\n                  and drove protease-dependent cell-state transitions in C3H\/10T1\/2 fibroblasts. The scFv\u2013NRR module also functioned as a protease-activated pro-antibody that conditionally inhibited DLL4-dependent signaling and ligand-independent activation of mutant NOTCH1 in the T cell acute lymphoblastic leukemia cell line HPB-ALL. Together, these results establish ProNotch as a modular platform for engineering protease-responsive cells and demonstrate that its regulatory module can be extended to a soluble, conditionally activated inhibitor of NOTCH1 signaling.\n                <\/jats:p>","DOI":"10.64898\/2026.06.01.728985","type":"posted-content","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T05:05:19Z","timestamp":1780549519000},"source":"Crossref","is-referenced-by-count":0,"title":["ProNotch converts extracellular protease activity into programmable transcriptional outputs"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6692-5899","authenticated-orcid":false,"given":"Jeremy C.","family":"Tran","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering, Biological Design Center, and Center for Multiscale Translational and Mechanobiology, Boston University, Boston, MA 02138"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4379-677X","authenticated-orcid":false,"given":"Christopher J.","family":"Kuffner","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Biological Design Center, and Center for Multiscale Translational and Mechanobiology, Boston University, Boston, MA 02138"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9117-4379","authenticated-orcid":false,"given":"Aiden C.","family":"Reilly","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Biological Design Center, and Center for Multiscale Translational and Mechanobiology, Boston University, Boston, MA 02138"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9712-328X","authenticated-orcid":false,"given":"Quan","family":"Le","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Biological Design Center, and Center for Multiscale Translational and Mechanobiology, Boston University, Boston, MA 02138"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3508-1915","authenticated-orcid":false,"given":"John T.","family":"Ngo","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Biological Design Center, and Center for Multiscale Translational and Mechanobiology, Boston University, Boston, MA 02138"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026060622050450000_2026.06.01.728985v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/J.JBC.2024.107347"},{"key":"2026060622050450000_2026.06.01.728985v1.2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1742-4658.2010.07920.x"},{"key":"2026060622050450000_2026.06.01.728985v1.3","doi-asserted-by":"publisher","DOI":"10.1111\/febs.15227"},{"key":"2026060622050450000_2026.06.01.728985v1.4","doi-asserted-by":"crossref","first-page":"1743","DOI":"10.1016\/j.drudis.2022.03.011","article-title":"Exploiting protease activation for therapy","volume":"27","year":"2022","journal-title":"Drug Discov. 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