Untersuchung der Substratspezifizität und Aktivitätsregulierung der Rhomboid Proteasen
Biochemie
Zusammenfassung der Projektergebnisse
Proteases cleave protein and polypeptide substrates in a highly regulated manner, and dysregulation can lead to various diseases. The development of chemical methods and tools to study protease selectivity and function has supported basic research and drug development. Activity-based protein profiling, developed since the start of the millenium, makes use of covalent chemical probes and has many different applications within protease research. Various families of intramembrane proteases exist: these were originally thought to be very peculiar, but turned out to occur in virtually all sequenced organisms. Rhomboid proteases, which are intramembrane serine proteases, are amongst the most well conserved membrane proteins and intramembrane proteases. Their function is diverse, but their role in various organisms is unknown and their druggability is still unclear. The aim of this project was to develop methods to study rhomboid activity in a membrane environment, to develop proteomics-based methods for identification of substrate specificity information and to use this information for the synthesis of selective chemical probes. We have successfully developed a protocol to enable the reconstitution of bacterial rhomboid proteases in liposomes. The resulting liposomes have a size distribution corresponding to large unilamellar vesicles and contain active rhomboid proteases. Transformation into giant unilamellar vesicles enabled visualization of rhomboid activity using a combination of fluorescent microscopy and activity-based probes, suggesting that chemical probes can be used for future imaging studies of rhomboid proteases. To enable a simple and inexpensive method for protease substrate specificity profiling without making use of isotope labels, we developed a novel methodology that relies on “charged synchronized” proteome-derived peptide libraries and separation of cleaved sequences by a charged-based separation in a pipette-tip. The applicability of this method was demonstrated by using five different soluble proteases with different cleavage specificities. The resulting cleavage consensus sequences stemming from protease cleavage sites matched well with the reported substrate specificity. Interestingly, for cathepsin G, we identified a previously unnoticed preference for asparagine in the P1 position. Application of this method to rhomboid proteases resulted in much less cleavage sites, as expected for a protease with low activity and a substrate recognition that spans both primed and non primed sites. However, utilization of an inhibitor-treated control sample enabled us to filter out many false positives, resulting in a substrate specificity profile with commonly reported, but also novel features. We applied substrate specificity information for the design and synthesis of novel covalent chemical probes targeting proteases. Central in this design were the replacement of the scissile bond by an uncleavable peptide bond mimic and the incorporation of a photocrosslinker. Unexpectedly, proof-ofconcept studies on caspase-3 revealed that, although they inhibit the target protease upon irradiation, do not lead to covalent modification of the target protease. It turned out that the “reduced amide” peptide bond mimic leads, upon irradiation in presence of benzophenone, via oxidation to formation of a peptide aldehyde, which is a potent, but reversible inhibitor of caspase-3. We envisage that this type of activation of an otherwise inactive inhibitor may be utilized in the future for photopharmacology studies, as it would enable the inhibition at a specific time and location.
Projektbezogene Publikationen (Auswahl)
- (2019) Short Peptides with Uncleavable Peptide Bond Mimetics as Photoactivatable Caspase-3 Inhibitors. Molecules 24 (1) 206
Van Kersavond, T.; Konopatzki, R.; Chakrabarty, S.; Blank-Landeshammer, B.; Sickmann, A.; Verhelst, S. H. L.
(Siehe online unter https://dx.doi.org/10.3390%2Fmolecules24010206) - (2015) Activity-based protein profiling of rhomboid proteases in liposomes. ChemBioChem, 16: 1616-1621
Wolf, E. V.; Seybolt, M.; Hadravova, R.; Strisovsky, K.; Verhelst, S. H. L.
(Siehe online unter https://doi.org/10.1002/cbic.201500213) - (2015) Chemical tools for the study of intramembrane proteases. ACS Chem. Biol., 10: 2423-2434
Nguyen, M.; Van Kersavond, T.; Verhelst, S. H. L.
(Siehe online unter https://doi.org/10.1021/acschembio.5b00693) - (2016) Detection of protease activity in cells and animals. Biochemical Biophysical Acta, 1864: 130-142
Verdoes, M.; Verhelst, S. H. L.
(Siehe online unter https://doi.org/10.1016/j.bbapap.2015.04.029) - (2016) Inhibitors of rhomboid proteases. Biochimie, 122:38-47
Wolf, E. V.; Verhelst, S. H. L.
(Siehe online unter https://doi.org/10.1016/j.biochi.2015.07.007) - (2017) Intramembrane proteases as drug targets. FEBS J., 284: 1489-1502
Verhelst, S. H. L.
(Siehe online unter https://doi.org/10.1111/febs.13979) - (2017) Synthesis and application of activity-based probes for proteases. Methods Mol. Biol., 1574: 255-266
Van Kersavond, T.; Nguyen, M.; Verhelst, S. H. L.
(Siehe online unter https://doi.org/10.1007/978-1-4939-6850-3_19) - (2018) Protease specificity profiling in a pipette tip using “charged-synchronized” proteome-derived peptide libraries, J. Proteome Res., 17: 1923-1933
Nguyen, M. T. N.; Shema, G.; Zahedi, R. P.; Verhelst, S. H. L.
(Siehe online unter https://doi.org/10.1021/acs.jproteome.8b00004)
