Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 166

From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 166 10

the 1st Faculty of Medicine Charles University Kateřinská 32 Prague 121 08 and

the Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 Prague 128

the Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 Prague 128

the Department of Physics and Materials Engineering Tomas Bata University in Zlín Faculty of Technology nám T G Masaryka 5555 76001 Zlín Czech Republic

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Urban S., Lee J. R., and Freeman M. (2002) A family of rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands. EMBO J. 21, 4277–4286 PubMed PMC

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 PubMed

McQuibban G. A., Saurya S., and Freeman M. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541 PubMed

O'Donnell R. A., Hackett F., Howell S. A., Treeck M., Struck N., Krnajski Z., Withers-Martinez C., Gilberger T. W., and Blackman M. J. (2006) Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite. J. Cell Biol. 174, 1023–1033 PubMed PMC

Fleig L., Bergbold N., Sahasrabudhe P., Geiger B., Kaltak L., and Lemberg M. K. (2012) Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of membrane proteins. Mol. Cell 47, 558–569 PubMed

Riestra A. M., Gandhi S., Sweredoski M. J., Moradian A., Hess S., Urban S., and Johnson P. J. (2015) A Trichomonas vaginalis rhomboid protease and its substrate modulate parasite attachment and cytolysis of host cells. PLoS Pathog. 11, e1005294. PubMed PMC

Etheridge S. L., Brooke M. A., Kelsell D. P., and Blaydon D. C. (2013) Rhomboid proteins: a role in keratinocyte proliferation and cancer. Cell Tissue Res. 351, 301–307 PubMed

Chan E. Y., and McQuibban G. A. (2013) The mitochondrial rhomboid protease: its rise from obscurity to the pinnacle of disease-relevant genes. Biochim. Biophys. Acta 1828, 2916–2925 PubMed

Song W., Liu W., Zhao H., Li S., Guan X., Ying J., Zhang Y., Miao F., Zhang M., Ren X., Li X., Wu F., Zhao Y., Tian Y., Wu W., et al. (2015) Rhomboid domain containing 1 promotes colorectal cancer growth through activation of the EGFR signalling pathway. Nat. Commun. 6, 8022. PubMed PMC

Strisovsky K. (2016) Why cells need intramembrane proteases: a mechanistic perspective. FEBS J. 283, 1837–1845 PubMed

Pierrat O. A., Strisovsky K., Christova Y., Large J., Ansell K., Bouloc N., Smiljanic E., and Freeman M. (2011) Monocyclic β-lactams are selective, mechanism-based inhibitors of rhomboid intramembrane proteases. ACS Chem. Biol. 6, 325–335 PubMed PMC

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 PubMed PMC

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 PubMed PMC

Simeonov A., Jadhav A., Thomas C. J., Wang Y., Huang R., Southall N. T., Shinn P., Smith J., Austin C. P., Auld D. S., and Inglese J. (2008) Fluorescence spectroscopic profiling of compound libraries. J. Med. Chem. 51, 2363–2371 PubMed

Vosyka O., Vinothkumar K. R., Wolf E. V., Brouwer A. J., Liskamp R. M., and Verhelst S. H. (2013) Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay. Proc. Natl. Acad. Sci. U.S.A. 110, 2472–2477 PubMed PMC

Wolf E. V., Zeißler A., Vosyka O., Zeiler E., Sieber S., and Verhelst S. H. (2013) A new class of rhomboid protease inhibitors discovered by activity-based fluorescence polarization. PLoS ONE 8, e72307. PubMed PMC

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 PubMed

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 PubMed PMC

Zoll S., Stanchev S., Began J., Skerle J., Lepšik M., Peclinovská 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 PubMed PMC

VanAken T., Foxall-VanAken S., Castleman S., and Ferguson-Miller S. (1986) Alkyl glycoside detergents: synthesis and applications to the study of membrane proteins. Methods Enzymol. 125, 27–35 PubMed

Kamp F., Winkler E., Trambauer J., Ebke A., Fluhrer R., and Steiner H. (2015) Intramembrane proteolysis of β-amyloid precursor protein by γ-secretase is an unusually slow process. Biophys. J. 108, 1229–1237 PubMed PMC

Scheel G., Acevedo E., Conzelmann E., Nehrkorn H., and Sandhoff K. (1982) Model for the interaction of membrane-bound substrates and enzymes: hydrolysis of ganglioside GD1a by sialidase of neuronal membranes isolated from calf brain. Eur. J. Biochem. 127, 245–253 PubMed

Parry G., Palmer D. N., and Williams D. J. (1976) Ligand partitioning into membranes: its significance in determining Km and Ks values for cytochrome P-450 and other membrane bound receptors and enzymes. FEBS Lett. 67, 123–129 PubMed

Rath A., and Deber C. M. (2013) Design of transmembrane peptides: coping with sticky situations. Methods Mol. Biol. 1063, 197–210 PubMed

Wolf E. V., Zeissler A., and Verhelst S. H. (2015) Inhibitor fingerprinting of rhomboid proteases by activity-based protein profiling reveals inhibitor selectivity and rhomboid autoprocessing. ACS Chem. Biol. 10, 2325–2333 PubMed

Haedke U., Küttler E. V., Vosyka O., Yang Y., and Verhelst S. H. (2013) Tuning probe selectivity for chemical proteomics applications. Curr. Opin. Chem. Biol. 17, 102–109 PubMed

Lemberg M. K., Menendez J., Misik A., Garcia M., Koth C. M., and Freeman M. (2005) Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases. EMBO J. 24, 464–472 PubMed PMC

Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., and Pease L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 PubMed

Gibson D. G., Young L., Chuang R. Y., Venter J. C., Hutchison C. A. 3rd, and Smith H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 PubMed

Miroux B., and Walker J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 PubMed

Urbani A., and Warne T. (2005) A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research. Anal. Biochem. 336, 117–124 PubMed

Solínová V., Kasicka V., Koval D., Barth T., Ciencialová A., and Záková L. (2004) Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 808, 75–82 PubMed

Fenyo D., Wang Q., DeGrasse J. A., Padovan J. C., Cadene M., and Chait B. T. (2007) MALDI sample preparation: the ultra thin layer method. J. Vis. Exp. 192, 10.3791/192 PubMed DOI PMC

Nomenclature Committee of the International Union of Biochemistry (1979) Units of enzyme-activity, recommendations 1978. Eur. J. Biochem. 97, 319–320

Nomenclature Committee of the International Union of Biochemistry (1983) Symbolism and terminology in enzyme-kinetics, recommendations 1981. Biochem. J. 213, 561–571 PubMed PMC

Sreerama N., and Woody R. W. (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 287, 252–260 PubMed

Provencher S. W., and Glöckner J. (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20, 33–37 PubMed

Mongay C., and Cerda V. (1974) Britton-Robinson buffer of known ionic-strength. Anal. Chim. 64, 409–412

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