Abstract
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.
Acknowledgments
Funding: M.J.L. gratefully acknowledges supported by grant MOP-93557, Funder Id: 10.13039/501100000030 from the Canadian Institutes of Health Research, Neuroscience and Mental Health Institute (Brad Mates Foundation), Parkinson Alberta, and the Department of Biochemistry, University of Alberta. M.J.L. was supported in part by funding from Alberta Innovates Health Solutions and the Parkinson’s Society of Canada. J.Y. acknowledges funding by a CSC scholarship. S.V. was supported in part by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin and the Bundesministerium für Bildung und Forschung. A.J.O. gratefully acknowledges financial support from UC San Diego Skaggs School of Pharmacy and Pharmaceutical Science. H.S.Y. gratefully acknowledges support from the Heart and Stroke Foundation of Canada.
Conflict of interest statement: The authors declare that they have no conflicts of interest concerning the contents of this article.
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2018-0255).
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