Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases

Abstract

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

Probing substrate recognition of bacterial lipoprotein signal peptidase using FRET reporters

“…As the DDM concentration exceeded 0.5%, the apparent enzyme activity decreased and is presumably due to an increase in empty micelles and the relative decrease in accessible substrate. Our results are well correlated with a previous study on a transmembrane rhomboid protease that showed similar trends in activity with increases in DDM . In fact, in agreement with Tichá et al ., the slope of the Log–Log plot at high DDM concentration (Fig.…”

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

“…Bacillus subtilis genome encodes two rhomboid protease genes, ydcA and yqgP [also known as gluP (Mesak et al , )]. No proteolytic activity has been detected for YdcA so far (Urban et al , ; Lemberg et al , ), while YqgP is a commonly used model rhomboid protease cleaving a number of synthetic substrates (Lemberg et al , ; Urban & Wolfe, ; Ticha et al , ,b). YqgP has homologs in a number of Gram‐positive bacteria, including Bacilli, but also Staphylococci, Listeriae and others (http://www.ebi.ac.uk/interpro/protein/P54493/similar-proteins), and thus represents an attractive system to explore the cell biology and functions of bacterial rhomboid proteases.…”

Involvement of a conserved GFG motif region in substrate binding by RseP, an Escherichia coliS2P protease

“…Obviously structural analysis of the RseP–substrate complex will be needed to understand the molecular details of the interaction between RseP and a substrate. In addition, kinetic analysis using purified RseP and substrates in solubilized and reconstituted systems as conducted for other I‐CLiPs (Strisovsky et al ., ; Dickey et al ., ; Kamp et al ., ; Bolduc et al ., , Tichá et al ., ) will be useful for functional characterization of the two substrate‐binding sites. Such analyses would provide an important basis to elucidate the mechanisms underlying specific substrate recognition and cleavage by RseP and other S2P proteases.…”