Final Report Summary - RHOMBOID SUBSTRATES (Substrate specificity, mechanism and biological roles of rhomboid intramembrane proteases.)
More specifically, project work packages (WP) comprised:
I. Identification of molecular and biological functions of selected bacterial rhomboids
II. Understanding of molecular basis of rhomboid substrate specificity and mechanism
III. Identification of rhomboid substrates by quantitative proteomics
IV. Development of rhomboid inhibitors
Over the four years of the duration of the grant we have made progress on all four fronts, which resulted in 15 scientific publications in international peer-reviewed journals or monographs. Specifically, we have found that the E.coli rhomboid protease functions as an unprecedented membrane protein topology quality control protease, and the rhomboid protease in B.subtilis acts as a component of membrane protein quality control as well.
We have developed a quantitative proteomics platform for identification of rhomboid protease substrates, and applied it to both the bacterial rhomboid proteases mentioned above as well as to mammalian rhomboids. We have thus identified a substrate repertoire of human rhomboid protease RHBDL2, which implicates it in epithelial homeostasis.
We have uncovered the basic principles of substrate recognition by rhomboid proteases and solved the first ever structure of a complex of an intramembrane protease with substrate-derived peptides. This enabled us to devise novel, highly sensitive and versatile activity assays, and, more importantly, led us to discover a new class of rhomboid protease inhibitors that are potent and selective and have a clear rationale of how to modify their selectivity. These compounds constitute the first practically applicable tools for cell biology of rhomboid proteases as well as pharmacologically compliant compounds for future drug discovery efforts aimed at rhomboid proteases, yielding results that might be practically useful for the whole community.