Engineering subtilisin proteases that specifically degrade active RAS

被引:17
作者
Chen, Yingwei [1 ]
Toth, Eric A. [2 ,3 ,4 ]
Ruan, Biao [1 ]
Choi, Eun Jung [1 ]
Simmerman, Richard [1 ]
Chen, Yihong [2 ]
He, Yanan [2 ]
Wang, Ruixue [2 ]
Godoy-Ruiz, Raquel [2 ,4 ,5 ]
King, Harlan [2 ,6 ,7 ]
Custer, Gregory [2 ,8 ]
Gallagher, D. Travis [2 ,6 ,7 ]
Rozak, David A. [9 ]
Solomon, Melani [2 ,8 ]
Muro, Silvia [2 ,8 ,10 ,11 ]
Weber, David J. [2 ,3 ,4 ,5 ]
Orban, John [2 ,12 ]
Fuerst, Thomas R. [2 ,13 ]
Bryan, Philip N. [1 ,2 ]
机构
[1] Potomac Affin Prot, North Potomac, MD 20878 USA
[2] Inst Biosci & Biotechnol Res, Rockville, MD 20850 USA
[3] Univ Maryland, Sch Med, Marlene & Stewart Greenebaum Canc Ctr, Baltimore, MD 21201 USA
[4] Ctr Biomol Therapeut, Rockville, MD USA
[5] Univ Maryland, Sch Med, Dept Biochem & Mol Biol, Baltimore, MD 21201 USA
[6] NIST, Rockville, MD USA
[7] Univ Maryland, Rockville, MD USA
[8] Univ Maryland, Dept Bioengn, College Pk, MD 20742 USA
[9] US Army, Res Inst Infect Dis, Unified Culture Collect, Ft Detrick, MD USA
[10] Barcelona Inst Sci & Technol, Inst Bioengn Catalonia, Barcelona, Spain
[11] Barcelona Inst Sci & Technol, Inst Catalonia Res & Adv Studies, Barcelona, Spain
[12] Univ Maryland, Dept Chem & Biochem, College Pk, MD 20742 USA
[13] Univ Maryland, Dept Cell Biol & Mol Genet, College Pk, MD 20742 USA
关键词
CRYSTAL-STRUCTURE; STRUCTURAL BASIS; SUBSTRATE-SPECIFICITY; MACROMOLECULAR STRUCTURES; FOLDING REACTION; BINDING MODULE; EGLIN-C; PROTEIN; COMPLEX; MUTANT;
D O I
10.1038/s42003-021-01818-7
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
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. Chen et al. describe a rational design of subtilisin mutants that degrade active RAS by cleaving a conserved sequence in switch 2. They further modified the active site to be dependent on a cofactor to generate high target specificity. Proteases engineered to cleave this region degraded RAS in vitro and in cells with a promise of adaptability for other target proteins too.
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页数:13
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