Skip to main content
European Commission logo
français français
CORDIS - Résultats de la recherche de l’UE
CORDIS
CORDIS Web 30th anniversary CORDIS Web 30th anniversary
Contenu archivé le 2024-06-18

Substrate specificity, mechanism and biological roles of rhomboid intramembrane proteases.

Final Report Summary - RHOMBOID SUBSTRATES (Substrate specificity, mechanism and biological roles of rhomboid intramembrane proteases.)

Rhomboid intramembrane proteases are evolutionarily widespread. Their functions in model insect, worm, yeast and protozoan species are important, but in most organisms including mammals and bacteria are unknown. The key to the understanding of rhomboid functions are their natural substrates, but it has been unclear how these are selected and recognized and methods for substrate identification have been lacking. This was limiting progress in the field, and the present project addressed precisely this knowledge gap. This project aimed to discover the biological roles of highly conserved rhomboids by identifying their natural substrates using a combination of advanced substrate specificity analysis, quantitative proteomics and genetics. We have developed a platform of proteomic approaches in bacteria and mammalian cells to identify rhomboid protease substrates and used it to uncover the biological function of rhomboid proteases in human epithelial cells and in model gram positive and gram negative bacteria. We have gained a deep mechanistic and structural insight into rhomboid protease function, which has helped us design new activity assays and novel rhomboid protease inhibitors of unprecedented potency and selectivity, which might serve as lead class of compounds in future drug discovery efforts.

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.