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.

FIGURE 1.

Human mSH superfamily. A, dendrogram showing all 116 members of the mSH family in humans. Branch length depicts sequence relatedness based on a sequence alignment anchored around the serine nucleophiles. The enzyme names are colored according to their degree of characterization, with characterized enzymes shown in black and uncharacterized enzymes shown in blue. B, sequence-derived evidence that most mSHs, regardless of their degree of annotation, perform unique and conserved functions in mammals. The scatter plot shows the 116 mSHs arranged according to the percent sequence identity for their nearest human mSH neighbor (homology, y axis) and mouse ortholog (x axis). Proteins appearing in the lower right-hand corner are both unique within the human proteome (as they display low sequence identity with their nearest human neighbor) and well conserved across mammalian species (as their mouse and human orthologs display strong sequence conservation).

FIGURE 2.

Platforms and probes for ABPP of SHs. A, common ABPP platforms. In the upper panel, an activity-based probe conjugated to a fluorophore (right) or biotin (left) tag is shown labeling a proteome. Probe-labeled proteins can then be visualized directly via SDS-PAGE and fluorescent gel scanning (fluorophore probes) or enriched with avidin and identified by LC/LC-MS/MS (ABPP-MudPIT; biotin probes). The lower panel shows both of these techniques being applied to the characterization of inhibitors (competitive ABPP). B, covalent modification of the active-site serine nucleophile of SHs by a FP activity-based probe. The red stars indicate a tag, such as a fluorophore or biotin.