Abstract
Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.
Highlights
Proteases catalyze the hydrolysis of peptide bonds in proteins and are involved in digestive as well as regulatory processes
While most proteases are soluble, a small fraction is membrane-embedded [1]. These intramembrane proteases differ from soluble proteases in a variety of aspects: They are composed of a number of transmembrane domains (TMDs) which harbor the catalytic residues with their active sites buried several Ainto the membrane
In view of previous work of the Cravatt laboratory [25], we expected that fluorescent rhomboid activity-based probes (ABPs) would be suitable for the development of a gel-free FluoPol ABPP screening method
Summary
Proteases catalyze the hydrolysis of peptide bonds in proteins and are involved in digestive as well as regulatory processes. While most proteases are soluble, a small fraction is membrane-embedded [1]. These intramembrane proteases differ from soluble proteases in a variety of aspects: They are composed of a number of transmembrane domains (TMDs) which harbor the catalytic residues with their active sites buried several Ainto the membrane. Their substrates are transmembrane proteins that reside inside the membrane in a dormant form. It is not surprising that intramembrane proteases are involved in various signaling pathways [1,2,3]
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