Activity-based protein profiling of the human rhomboid proteases for inhibitor discovery and enzyme characterization

Oberlin College, Oberlin OH

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Abstract

It has been estimated that there could be more than one million different protein species in the human body. Our understanding of the biological roles of these various species is still limited to a small subset of proteins. Hydrolases, proteins that cleave chemical bonds using water, represent one of the largest classes of enzymes in the human body and play essential roles in numerous physiological pathways ranging from metabolism to nervous system signaling. Despite the prevalence and importance of this enzyme class, the biological functions of many hydrolases are still not well-characterized. Among the hydrolases with limited characterization, rhomboid proteases, which perform hydrolysis chemistry within the hydrophobic environment of biological membranes, have generated notable interest. Mutation and dysregulation of these enzymes in neurodegenerative diseases and multiple types of cancer motivates the development of an enhanced understanding of their physiological roles and an investigation of their potential to serve as therapeutic targets; however, methods to conduct these studies are currently lacking. Activity-based protein profiling (ABPP) technology, in which active site-directed chemical probes are used to detect enzyme activity, has facilitated the characterization of dozens of serine hydrolases. As members of this superfamily of enzymes, the human rhomboid proteases (RHBDL1, RHBDL2, RHBDL3, RHBDL4, PARL) represent promising candidates for study by ABPP methods, though suitable probes are needed to perform these studies. This proposal aims to (1) convert previously disclosed rhomboid protease inhibitors into new activity-based probes for the rhomboid proteases and other serine hydrolases, (2) develop an optimized b-lactone probe to establish a robust ABPP assay to study inhibition and proteolytic processing of RHBDL2, and (3) study the potential influence of the large aqueous-exposed N-terminal domains of RHBDL1 and RHBDL3 on the activity of these enzymes. These studies will empower ongoing efforts to investigate the biological roles of the human rhomboid proteases and, ultimately, their potential to serve as therapeutic targets. Importantly, the proposed project will provide an opportunity to train Oberlin undergraduate researchers in contemporary chemical biology methods that incorporate techniques from synthetic chemistry, biochemistry, and molecular biology. Research students at Oberlin will learn theory and advanced laboratory skills not otherwise covered in an undergraduate classroom or teaching laboratory setting while also further developing their ability to analyze and present scientific data. Collectively, these activities will enrich the research environment at Oberlin College and help prepare the next generation of researchers in interdisciplinary molecular science.