Engineering subtilisin proteases that specifically degrade active RAS.

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TitleEngineering subtilisin proteases that specifically degrade active RAS.
Publication TypeJournal Article
Year of Publication2021
AuthorsChen, Y, Toth, EA, Ruan, B, Choi, EJung, Simmerman, R, Chen, Y, He, Y, Wang, R, Godoy-Ruiz, R, King, H, Custer, G, D Gallagher, T, Rozak, DA, Solomon, M, Muro, S, Weber, DJ, Orban, J, Fuerst, TR, Bryan, PN
JournalCommun Biol
Volume4
Issue1
Pagination299
Date Published2021 Mar 05
ISSN2399-3642
Abstract

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

DOI10.1038/s42003-021-01818-7
Alternate JournalCommun Biol
PubMed ID33674772
PubMed Central IDPMC7935941
Grant ListR01 GM062154 / GM / NIGMS NIH HHS / United States
R01GM062154 / / U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) /
R44GM126676 / / U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) /
R44GM103389 / / U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) /
R44 GM126676 / GM / NIGMS NIH HHS / United States
R44 GM103389 / GM / NIGMS NIH HHS / United States
R44CA163403 / / U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI) /
R44 CA163403 / CA / NCI NIH HHS / United States