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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

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Volume 399, Issue 12


An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases

Elena Arutyunova
  • Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
  • Other articles by this author:
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/ Zhenze Jiang
  • Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
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/ Jian Yang
  • KU Leuven – University of Leuven, Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, Herestraat 49 Box 802, B-3000 Leuven, Belgium
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/ Ayodeji N. Kulepa
  • Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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/ Howard S. Young
  • Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
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/ Steven Verhelst
  • KU Leuven – University of Leuven, Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, Herestraat 49 Box 802, B-3000 Leuven, Belgium
  • Leibniz Institute for Analytical Sciences ISAS, Otto-Hahn-Str. 6b, D-44227 Dortmund, Germany
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/ Anthony J. O’Donoghue
  • Corresponding author
  • Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
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/ M. Joanne Lemieux
  • Corresponding author
  • Department of Biochemistry, Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton T6G 2R3, Alberta, Canada
  • Email
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Published Online: 2018-11-13 | DOI: https://doi.org/10.1515/hsz-2018-0255


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.

This article offers supplementary material which is provided at the end of the article.

Keywords: bicelles; FRET; GlpG; intramembrane protease; kinetics; membrane protein; peptide; rhomboid protease; serine protease


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About the article

aElena Arutyunova and Zhenze Jiang: These authors contributed equally to this work.

Received: 2018-05-17

Accepted: 2018-07-09

Published Online: 2018-11-13

Published in Print: 2018-11-27

Conflict of interest statement: The authors declare that they have no conflicts of interest concerning the contents of this article.

Citation Information: Biological Chemistry, Volume 399, Issue 12, Pages 1389–1397, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2018-0255.

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