Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes

Abstract

Although activity-based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity-based imaging of rhomboid proteases in membrane environments.

Inhibitors of rhomboid proteases

“…For rhomboids, several activity-based probes have been reported [35,37,45]. They enable a variety of different detection methods including gel-based analysis [35,37,45], fluorescent polarization in 96-well plate format [35], and fluorescent microscopy [46]. The structures and applications of rhomboid ABPs will be discussed in more detail in Section 4.…”

“…However, this study showed that the identification of inhibitors is not affected by the environment, confirming that micelle-solubilized rhomboids are a suitable model system. Furthermore, it was shown that fluorescent ABPs can be used to visualize rhomboids in giant unilamellar vesicles by fluorescence microscopy [46].…”

“…Recently, ABPs were also applied in the labeling of rhomboid proteases in liposomes [46]. In contrast to detergent micelles, liposomes embed rhomboids within a lipid bilayer.…”

Discovery and Biological Evaluation of Potent and Selective N-Methylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases

“…The selectivity of the compounds for a variety of rhomboids and off-target effects against human serine hydrolases were tested using activity-based probe approaches. 36,37 This effort provided submicromolar, partially reversible, and selective rhomboid protease inhibitors based on a novel and modifiable scaffold.…”

“…Mass spectrometry and mutagenesis were used to establish the reaction mechanism of rhomboid inhibition by saccharins, and the in vivo potency was determined in Escherichia coli . The selectivity of the compounds for a variety of rhomboids and off-target effects against human serine hydrolases were tested using activity-based probe approaches. , This effort provided submicromolar, partially reversible, and selective rhomboid protease inhibitors based on a novel and modifiable scaffold.…”