期刊
BIOLOGICAL CHEMISTRY
卷 399, 期 12, 页码 1389-1397出版社
WALTER DE GRUYTER GMBH
DOI: 10.1515/hsz-2018-0255
关键词
bicelles; FRET; GlpG; intramembrane protease; kinetics; membrane protein; peptide; rhomboid protease; serine protease
资金
- Canadian Institutes of Health Research, Neuroscience and Mental Health Institute [MOP-93557]
- Parkinson Alberta
- Department of Biochemistry, University of Alberta
- Alberta Innovates Health Solutions
- Parkinson's Society of Canada
- CSC scholarship
- Ministerium fur Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen
- Senatsverwaltung fur Wirtschaft, Technologie und Forschung des Landes Berlin
- Bundesministerium fur Bildung und Forschung
- UC San Diego Skaggs School of Pharmacy and Pharmaceutical Science
- Heart and Stroke Foundation of Canada
Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据