Analysis of the substrate specificity and activity regulation of rhomboid proteases

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Intramembrane proteases are unusual enzymes that hydrolyze their substrates in the hydrophobic environment of the membrane. Generally, the substrates are bound to the membrane in a dormant form, which upon cleavage lead to a specific biological response. The rhomboids form the family of intramembrane serine proteases. Structurally, they are the best characterized intramembrane proteases and they therefore form a paradigm for intramembrane proteolysis. Interestingly, genes coding for rhomboids have been found in virtually all sequenced organisms, but the functional role of most of them remains unelucidated. From the examples that are known, it appears that the biological function of rhomboids is quite diverse, ranging from cellular communication to host cell invasion by certain parasites. There are several key questions in rhomboid research that remain unanswered: How does rhomboid choose its substrates? How does the substrate enter the active site? Can rhomboids be used as drug targets? Can selective inhibitors be designed? And how is rhomboid activity regulated? The specific aims of the project described in this proposal deal with several of these questions. Previous contradicting clues about the substrate specificity were obtained from the study of specific protein substrates. We will reveal the substrate specificity of rhomboids by an unbiased, proteomics-based approach. Making use of proteome-derived peptide libraries, we will identify scissile bonds of different rhomboids by mass spectrometry-based methods. The resulting cleavage sites will be analyzed in order to derive a consensus sequence for each individual rhomboid. The resulting information will them be used to design new reporter substrates and novel peptide-based inhibitors, which will be analyzed for their selectivity and their site of interaction with the rhomboid structure. Although most proteases are synthesized as inactive zymogens, rhomboids are translated in their active from. There are some indications that the membrane environment influences substrate cleavage. To gain more insight into this, we will express and purify rhomboids from different prokaryotic and eukaryotic species and reconstitute them in liposomes with different lipid compositions. Using our recently developed rhomboid ABPs, we have the ideal tools in hand to measure the activity state of the rhomboids in these different environments. Overall, this proposal will yield valuable information about the activity regulation and the substrate specificity of rhomboids, and may form the foundation for future rhomboid drug development.