Activity-based Proteomics of Enzyme Superfamilies: Serine Hydrolases as a Case Study

Abstract

Genome sequencing projects have uncovered thousands of uncharacterized enzymes in eukaryotic and prokaryotic organisms. Deciphering the physiological functions of enzymes requires tools to profile and perturb their activities in native biological systems. Activity-based protein profiling has emerged as a powerful chemoproteomic strategy to achieve these objectives through the use of chemical probes that target large swaths of enzymes that share active-site features. Here, we review activity-based protein profiling and its implementation to annotate the enzymatic proteome, with particular attention given to probes that target serine hydrolases, a diverse superfamily of enzymes replete with many uncharacterized members.Complete genome sequences have revolutionized our view of living systems. The number of genes possessed by organisms ranging from bacteria to yeast to humans is now more or less confidently assigned and has provided a framework for understanding complex cellular and physiological processes at a biochemical level. This framework is, however, plagued by huge knowledge gaps represented, perhaps most notably, by a daunting number of uncharacterized gene products. These include many predicted proteins that lack discernible sequence homology to other proteins of known function, as well as expansive protein families, of which only a modest subset of members have been assigned biochemical activities (1, 2).Working under the Darwinian assumption that every protein has evolved to perform a unique function that ultimately benefits the host organism, it follows that significant gaps in our knowledge of the proteome imperil ongoing computational and experimental attempts to build molecular networks that explain higher order life processes. This problem is accentuated by the realization that among human protein families containing many poorly characterized members are the fundamental components of signal transduction (receptors, ion channels), gene regulation (transcription factors), and metabolic (enzymes, transporters) pathways. Ongoing and future "systems biology" endeavors would thus greatly benefit from new technologies that facilitate assignment of protein function on a global scale. These technologies can take the form of methods that map fundamental features of protein behavior, including intermolecular (e.g. gene-gene, protein-protein, protein-DNA, protein-metabolite) interactions (3, 4), cellular and subcellular localization (5), post-translational modification state (6), and biochemical activities (7-9). In this minireview, we will focus on the last category in our discussion of the chemoproteomic method activity-based protein profiling (ABPP), 2 which aims to globally characterize the functional state of enzymes in native biological systems (8, 9). Our goal is to showcase how, in little more than a decade, ABPP has developed into a versatile platform for enzyme annotation in the genomic era. Enzyme Analysis by ABPP: Serine Hydrolases as a Case StudyABPP has been successfully applied to many enzyme cla...

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