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

Abstract

Rhomboids 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 the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid 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 from Providencia 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.

Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation

“…While helical, lipid-embedded substrate TM segments are thought to unfold into the active site via the membrane-embedded lateral gate (Cho et al, 2016), it is conceivable that for RHBDL4 the surface-active site opening allows flexible loops of ERAD-L substrates to enter the active site from the ER lumen. Accordantly, we and others observed rhomboid cleavage within ectodomains and loops of membrane proteins (Fleig et al, 2012;Knopf et al, submitted;Maegawa et al, 2007;Paschkowsky et al, 2018) and detergent solubilized rhomboids are known to cleave in vitro soluble model substrates (Arutyunova et al, 2018;Maegawa et al, 2005;Wang et al, 2006). Here, we now show cleavage of a soluble ERAD-L substrate in a physiological context.…”

Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

“…To further evaluate the cleavage of other known substrates of PARL, we adopted a more facile system to examine cleavage of multiple substrates, and generated internally quenched (IQ) peptide substrates (25,(36)(37)(38) based on the amino acids flanking the PARL cleavage sites of PINK1 (39), PGAM5 (8), and Smac/Diablo (11). Kinetic analysis using both full-length and βtruncated PARL with IQ-PINK1 99-108 , IQ-PGAM5 [20][21][22][23][24][25][26][27][28][29] , and IQ-Smac/Diablo 51-60 peptide substrates was first performed in detergent, which revealed similar Michaelis-Menten kinetics for all peptides (Fig.…”

Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

“…To further evaluate the cleavage of other known substrates of PARL, we generated internally quenched (IQ) peptide substrates (Arutyunova et al, 2018;Lapek et al, 2019;Naing et al, 2018;Ticha et al, 2017a) based on the amino acids flanking the PARL cleavage sites of PINK1 (Deas et al, 2011), PGAM5 (Sekine et al, 2012), and Smac (Saita et al, 2017). Kinetic analysis using both full-length and -truncated PARL with IQ-PINK1 [99][100][101][102][103][104][105][106][107][108] , IQ-PGAM5 [20][21][22][23][24][25][26][27][28][29] , and IQ-Smac 51-60 peptide substrates was first performed in detergent, which revealed similar Michaelis-Menten kinetics for all peptides (Fig 1F).…”