An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases

Abstract

AbstractRhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that theEscherichia colirhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and theHaemophilus influenzaerhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA fromProvidencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.