Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis: the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.

Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway signaled by caspase-1 and the noncanonical pathway regulated by mouse caspase-11 and human caspases-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors pro-interleukin-18 and pro-interleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1’–P4’—the C-terminal gasdermin D region adjacent to the cleavage site—influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1’–P4’ region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable to that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1’–P4’ region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11—i.e. caspases-4 and -5—are ruled by the same mechanism. We propose that P1’–P4’-based inhibitors could be exploited to specifically target inflammatory caspases.