Assay development for monitoring intramembrane (Rhomboid) serine protease substrate cleavage and inhibition

Rhomboid proteases are a recently discovered member of the unusual family of intramembrane proteases.They are conserved in all kingdoms of life and fulfill highly regulatory functions like EGF-receptor signaling, parasitic host cell invasion or quorum sensing in the pathogenic bacterium Providencia stuarti.Although they seem to be involved in a variety of processes in health and disease, little is known about their substrates and mechanism of action, partly due to the lack of suitable specific inhibitors and activity-based probes (ABP).We developed a mass spectrometry based assay that utilizes a natural protein substrate and used it to screen for inhibitors and activators of bacterial rhomboid proteases.This assay may also be applicable for rhomboids from other species, other intramembrane proteases and is shown to be suitable to determine rhomboid cleavage kinetics.We identified a range of inhibitors with IC 50 values in the low micromolar range and discovered an unusual mode of inhibitor binding using protein crystallization and determine their mechanism of action in biochemical studies.The new ABPs, identified in the screening are molecules that label active rhomboids but not their inactive counterparts and can for example be used to study regulatory mechanisms of protease activity.We used the ABPs to label endogenous E. coli rhomboid GlpG in cell lysates as well as expressed GlpG in vivo.This provides a powerful set of tools for functional cellular assays addressing the biological function of bacterial rhomboids, which is not known for many rhomboids.