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

  • Elena Arutyunova , Zhenze Jiang , Jian Yang , Ayodeji N. Kulepa , Howard S. Young , Steven Verhelst , Anthony J. O’Donoghue EMAIL logo und M. Joanne Lemieux EMAIL logo
Veröffentlicht/Copyright: 13. November 2018
Biological Chemistry
Aus der Zeitschrift Biological Chemistry Band 399 Heft 12

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.

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

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.

  1. 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).

Received: 2018-05-17
Accepted: 2018-07-09
Published Online: 2018-11-13
Published in Print: 2018-11-27

©2018 Walter de Gruyter GmbH, Berlin/Boston

Artikel in diesem Heft

  1. Frontmatter
  2. Highlight: Frontiers in Proteolysis
  3. Host cell-surface proteins as substrates of gingipains, the main proteases of Porphyromonas gingivalis
  4. A single domain antibody against the Cys- and His-rich domain of PCSK9 and evolocumab exhibit different inhibition mechanisms in humanized PCSK9 mice
  5. Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome
  6. An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases
  7. An alternative processing pathway of APP reveals two distinct cleavage modes for rhomboid protease RHBDL4
  8. Reviews
  9. Salivary peptide histatin 1 mediated cell adhesion: a possible role in mesenchymal-epithelial transition and in pathologies
  10. Modulation of dynamin function by small molecules
  11. Chemotherapeutic resistance: a nano-mechanical point of view
  12. Research Articles/Short Communications
  13. Protein Structure and Function
  14. Biochemical and kinetic properties of the complex Roco G-protein cycle
  15. Cell Biology and Signaling
  16. Aberrant expression of hsa_circ_0025036 in lung adenocarcinoma and its potential roles in regulating cell proliferation and apoptosis
https://doi.org/10.1515/hsz-2018-0255
Schlagwörter für diesen Artikel
bicelles; FRET; GlpG; intramembrane protease; kinetics; membrane protein; peptide; rhomboid protease; serine protease

Artikel in diesem Heft

  1. Frontmatter
  2. Highlight: Frontiers in Proteolysis
  3. Host cell-surface proteins as substrates of gingipains, the main proteases of Porphyromonas gingivalis
  4. A single domain antibody against the Cys- and His-rich domain of PCSK9 and evolocumab exhibit different inhibition mechanisms in humanized PCSK9 mice
  5. Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome
  6. An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases
  7. An alternative processing pathway of APP reveals two distinct cleavage modes for rhomboid protease RHBDL4
  8. Reviews
  9. Salivary peptide histatin 1 mediated cell adhesion: a possible role in mesenchymal-epithelial transition and in pathologies
  10. Modulation of dynamin function by small molecules
  11. Chemotherapeutic resistance: a nano-mechanical point of view
  12. Research Articles/Short Communications
  13. Protein Structure and Function
  14. Biochemical and kinetic properties of the complex Roco G-protein cycle
  15. Cell Biology and Signaling
  16. Aberrant expression of hsa_circ_0025036 in lung adenocarcinoma and its potential roles in regulating cell proliferation and apoptosis
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