Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

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


IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

Online
ISSN
1437-4315
See all formats and pricing
More options …
Volume 399, Issue 12

Issues

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:
  • De Gruyter OnlineGoogle Scholar
/ Zhenze Jiang
  • Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ 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
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Ayodeji N. Kulepa
  • 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:
  • De Gruyter OnlineGoogle Scholar
/ Howard S. Young
  • 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:
  • De Gruyter OnlineGoogle Scholar
/ 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
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ 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
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ 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
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
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.CrossrefPubMedGoogle 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.PubMedGoogle 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.CrossrefGoogle 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.PubMedGoogle 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.CrossrefGoogle 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.CrossrefPubMedGoogle 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.CrossrefPubMedGoogle 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.PubMedCrossrefGoogle Scholar

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

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

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

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

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

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

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

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

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

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

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

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

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

Export Citation

©2018 Walter de Gruyter GmbH, Berlin/Boston.Get Permission

Supplementary Article Materials

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

[1]
Hester A. Beard, Marta Barniol-Xicota, Jian Yang, and Steven H. L. Verhelst
ACS Chemical Biology, 2019
[2]
Ana-Nicoleta Bondar and M. Joanne Lemieux
Chemical Reviews, 2019, Volume 119, Number 9, Page 6162

Comments (0)

Please log in or register to comment.
Log in