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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

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Volume 399, Issue 12

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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
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Published Online: 2018-11-13 | DOI: https://doi.org/10.1515/hsz-2018-0255

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.

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

References

  • Arutyunova, E., Panwar, P., Skiba, P.M., Gale, N., Mak, M.W., and Lemieux, M.J. (2014). Allosteric regulation of rhomboid intramembrane proteolysis. EMBO J. 33, 1869–1881.Google Scholar

  • Arutyunova, E., Smithers, C.C., Corradi, V., Espiritu, A.C., Young, H.S., Tieleman, D.P., and Lemieux, M.J. (2016). Probing catalytic rate enhancement during intramembrane proteolysis. Biol. Chem. 397, 907–919.Google Scholar

  • Baker, R.P., Young, K., Feng, L., Shi, Y., and Urban, S. (2007). Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate. Proc. Natl. Acad. Sci. USA 104, 8257–8262.Google Scholar

  • Boulware, K.T., Jabaiah, A., and Daugherty, P.S. (2010). Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics. Biotechnol. Bioeng. 106, 339–346.Google Scholar

  • Brooks, C.L., Lazareno-Saez, C., Lamoureux, J.S., Mak, M.W., and Lemieux, M.J. (2011). Insights into substrate gating in H. influenzae rhomboid. J. Mol. Biol. 407, 687–697.Google Scholar

  • Cho, S., Dickey, S.W., and Urban, S. (2016). Crystal structures and inhibition kinetics reveal a two-stage catalytic mechanism with drug design implications for rhomboid proteolysis. Mol. Cell. 61, 329–340.Google Scholar

  • Debela, M., Magdolen, V., Schechter, N., Valachova, M., Lottspeich, F., Craik, C.S., Choe, Y., Bode, W., and Goettig, P. (2006). Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences. J. Biol. Chem. 281, 25678–25688.Google Scholar

  • Dickey, S.W., Baker, R.P., Cho, S., and Urban, S. (2013). Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity. Cell. 155, 1270–1281.Google Scholar

  • Drag, M. and Salvesen, G.S. (2010). Emerging principles in protease-based drug discovery. Nat. Rev. Drug Discov. 9, 690–701.Google Scholar

  • Durr, U.H., Gildenberg, M., and Ramamoorthy, A. (2012). The magic of bicelles lights up membrane protein structure. Chem. Rev. 112, 6054–6074.Google Scholar

  • Dusterhoft, S., Kunzel, U., and Freeman, M. (2017). Rhomboid proteases in human disease: mechanisms and future prospects. Biochim. Biophys. Acta 1864, 2200–2209.Google Scholar

  • Erez, E. and Bibi, E. (2009). Cleavage of a multispanning membrane protein by an intramembrane serine protease. Biochemistry 48, 12314–12322.Google Scholar

  • Goel, P., Jumpertz, T., Ticha, A., Ogorek, I., Mikles, D.C., Hubalek, M., Pietrzik, C.U., Strisovsky, K., Schmidt, B., and Weggen, S. (2018). Discovery and validation of 2-styryl substituted benzoxazin-4-ones as a novel scaffold for rhomboid protease inhibitors. Bioorg. Med. Chem. Lett. 28, 1417–1422.Google Scholar

  • Golde, T.E., Wolfe, M.S., and Greenbaum, D.C. (2009). Signal peptide peptidases: a family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins. Semin. Cell. Dev. Biol. 20, 225–230.Google Scholar

  • Gonzalez Flecha, F.L. (2017). Kinetic stability of membrane proteins. Biophys. Rev. 9, 563–572.Google Scholar

  • Goupil, L.S., Ivry, S.L., Hsieh, I., Suzuki, B.M., Craik, C.S., O’Donoghue, A.J., and McKerrow, J.H. (2016). Cysteine and aspartyl proteases contribute to protein digestion in the gut of freshwater planaria. PLoS Negl. Trop. Dis. 10, e0004893.Google Scholar

  • Harris, J.L., Backes, B.J., Leonetti, F., Mahrus, S., Ellman, J.A., and Craik, C.S. (2000). Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. USA 97, 7754–7759.Google Scholar

  • Hedstrom, L. (2002). An overview of serine proteases. Curr. Protoc. Protein Sci. Chapter 21, Unit 21.10.1–21.10.8.Google Scholar

  • Kasperkiewicz, P., Poreba, M., Snipas, S.J., Lin, S.J., Kirchhofer, D., Salvesen, G.S., and Drag, M. (2015). Design of a selective substrate and activity based probe for human neutrophil serine protease 4. PLoS One 10, e0132818.Google Scholar

  • Kateete, D.P., Katabazi, F.A., Okeng, A., Okee, M., Musinguzi, C., Asiimwe, B.B., Kyobe, S., Asiimwe, J., Boom, W.H., and Joloba, M.L. (2012). Rhomboids of Mycobacteria: characterization using an aarA mutant of Providencia stuartii and gene deletion in Mycobacterium smegmatis. PLoS One 7, e45741.Google Scholar

  • Langosch, D., Scharnagl, C., Steiner, H., and Lemberg, M.K. (2015). Understanding intramembrane proteolysis: from protein dynamics to reaction kinetics. Trends Biochem. Sci. 40, 318–327.Google Scholar

  • Lazareno-Saez, C., Arutyunova, E., Coquelle, N., and Lemieux, M.J. (2013). Domain swapping in the cytoplasmic domain of the Escherichia coli rhomboid protease. J. Mol. Biol. 425, 1127–1142.Google Scholar

  • Lee, J.R., Urban, S., Garvey, C.F., and Freeman, M. (2001). Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila. Cell 107, 161–171.Google Scholar

  • Lemieux, M.J., Reithmeier, R.A., and Wang, D.N. (2002). Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain. J. Struct. Biol. 137, 322–332.Google Scholar

  • Lemieux, M.J., Fischer, S.J., Cherney, M.M., Bateman, K.S., and James, M.N. (2007). The crystal structure of the rhomboid peptidase from Haemophilus influenzae provides insight into intramembrane proteolysis. Proc. Natl. Acad. Sci. USA 104, 750–754.Google Scholar

  • Li, H., Goh, B.N., Teh, W.K., Jiang, Z., Goh, J.P.Z., Goh, A., Wu, G., Hoon, S.S., Raida, M., Camattari, A., et al. (2018). Skin commensal Malassezia globosa secreted protease attenuates Staphylococcus aureus biofilm formation. J. Invest. Dermatol. 138, 1137–1145.Google Scholar

  • Lopez-Otin, C. and Bond, J.S. (2008). Proteases: multifunctional enzymes in life and disease. J. Biol. Chem. 283, 30433–30437.Google Scholar

  • Maegawa, S., Ito, K., and Akiyama, Y. (2005). Proteolytic action of GlpG, a rhomboid protease in the Escherichia coli cytoplasmic membrane. Biochemistry 44, 13543–13552.Google Scholar

  • Manolaridis, I., Kulkarni, K., Dodd, R.B., Ogasawara, S., Zhang, Z., Bineva, G., O’Reilly, N., Hanrahan, S.J., Thompson, A.J., Cronin, N., et al. (2013). Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1. Nature 504, 301–305.Google Scholar

  • Mesak, L.R., Mesak, F.M., and Dahl, M.K. (2004). Expression of a novel gene, gluP, is essential for normal Bacillus subtilis cell division and contributes to glucose export. BMC Microbiol. 4, 13.Google Scholar

  • Nagase, H., Fields, C.G., and Fields, G.B. (1994). Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). J. Biol. Chem. 269, 20952–20957.Google Scholar

  • Panigrahi, R., Arutyunova, E., Panwar, P., Gimpl, K., Keller, S., and Lemieux, M.J. (2016). Reversible Unfolding of rhomboid intramembrane proteases. Biophys. J. 110, 1379–1390.Google Scholar

  • Perona, J.J. and Craik, C.S. (1995). Structural basis of substrate specificity in the serine proteases. Protein. Sci. 4, 337–360.Google Scholar

  • Poreba, M., Salvesen, G.S., and Drag, M. (2017). Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity. Nat. Protoc. 12, 2189–2214.Google Scholar

  • Powers, J.C., Asgian, J.L., Ekici, O.D., and James, K.E. (2002). Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 102, 4639–4750.Google Scholar

  • Rather, P.N., Ding, X., Baca-DeLancey, R.R., and Siddiqui, S. (1999). Providencia stuartii genes activated by cell-to-cell signaling and identification of a gene required for production or activity of an extracellular factor. J. Bacteriol. 181, 7185–7191.Google Scholar

  • Rawson, R.B. (2013). The site-2 protease. Biochim. Biophys. Acta 1828, 2801–2807.Google Scholar

  • Russell, C.W., Richards, A.C., Chang, A.S., and Mulvey, M.A. (2017). The rhomboid protease GlpG promotes the persistence of extraintestinal pathogenic Escherichia coli within the gut. Infect. Immun. 85, 1–15.Google Scholar

  • Salisbury, C.M. and Ellman, J.A. (2006). Rapid identification of potent nonpeptidic serine protease inhibitors. Chem. Biochem. 7, 1034–1037.Google Scholar

  • Sherratt, A.R., Braganza, M.V., Nguyen, E., Ducat, T., and Goto, N.K. (2009). Insights into the effect of detergents on the full-length rhomboid protease from Pseudomonas aeruginosa and its cytosolic domain. Biochim. Biophys. Acta 1788, 2444–2453.Google Scholar

  • Stevenson, L.G., Strisovsky, K., Clemmer, K.M., Bhatt, S., Freeman, M., and Rather, P.N. (2007). Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase. Proc. Natl. Acad. Sci. USA 104, 1003–1008.Google Scholar

  • Strisovsky, K. (2013). Structural and mechanistic principles of intramembrane proteolysis – lessons from rhomboids. FEBS J. 280, 1579–1603.Google Scholar

  • Strisovsky, K., Sharpe, H.J., and Freeman, M. (2009). Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates. Mol. Cell. 36, 1048–1059.Google Scholar

  • Ticha, A., Stanchev, S., Skerle, J., Began, J., Ingr, M., Svehlova, K., Polovinkin, L., Ruzicka, M., Bednarova, L., Hadravova, R., et al. (2017). Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases. J. Biol. Chem. 292, 2703–2713.Google Scholar

  • Urban, S. and Dickey, S.W. (2011). The rhomboid protease family: a decade of progress on function and mechanism. Genome Biol. 12, 231.Google Scholar

  • Urban, S., Lee, J.R., and Freeman, M. (2001). Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107, 173–182.Google Scholar

  • Urban, S., Schlieper, D., and Freeman, M. (2002). Conservation of intramembrane proteolytic activity and substrate specificity in prokaryotic and eukaryotic rhomboids. Curr. Biol. 12, 1507–1512.Google Scholar

  • Vinothkumar, K.R. (2011). Structure of rhomboid protease in a lipid environment. J. Mol. Biol. 407, 232–247.Google Scholar

  • Wang, Y. and Ha, Y. (2007). Open-cap conformation of intramembrane protease GlpG. Proc. Natl. Acad. Sci. USA 104, 2098–2102.Google Scholar

  • Wang, Y., Zhang, Y., and Ha, Y. (2006). Crystal structure of a rhomboid family intramembrane protease. Nature 444, 1–5.Google Scholar

  • Wolfe, M.S. (2009). Intramembrane-cleaving proteases. J. Biol. Chem. 284, 13969–13973.Google Scholar

  • Xue, Y. and Ha, Y. (2012). Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate. J. Biol. Chem. 287, 3099–3107.Google Scholar

  • Yang, J., Barniol-Xicota, M., Nguyen, M.T.N., Ticha, A., Strisovsky, K., and Verhelst, S.H.L. (2018). Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases. Bioorg. Med. Chem. Lett. 28, 1423–1427.Google Scholar

  • Zoll, S., Stanchev, S., Began, J., Skerle, J., Lepsik, M., Peclinovska, L., Majer, P., and Strisovsky, K. (2014). Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures. EMBO J. 33, 2408–2421.Google Scholar

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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