Proteases are enzymes that hydrolyze the peptide bond of peptide substrates and proteins. Despite significant progress in recent years, one of the greatest challenges in the design and testing of substrates, inhibitors and activity‐based probes for proteolytic enzymes is achieving specificity toward only one enzyme. This specificity is particularly important if the enzyme is present with other enzymes with a similar catalytic mechanism and substrate specificity but completely different functionality. The cross‐reactivity of substrates, inhibitors and activity‐based probes with other enzymes can significantly impair or even prevent investigations of a target protease. In this review, we describe important concepts and the latest challenges, focusing mainly on peptide‐based substrate specificity techniques used to distinguish individual enzymes within major protease families.

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.

Neutrophils play a critical role in the first-line of defense against circulating pathogens and contain a wide array of granules that store antimicrobial proteins, with neutrophil serine proteases (NSPs) and defensins serving as crucial components. NSPs such as neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG) and neutrophil serine protease 4 (NSP4) exhibit distinct substrate specificities that underpin their critical roles in immune defense and inflammation [1]. After neutrophils are activated, they form and release neutrophil extracellular traps (NETs) consisting of decondensed chromatin and intracellular proteins through a process called NETosis, which leads to neutrophil death. Although NETosis is predominantly categorized as a suicidal process, several studies have suggested that neutrophils remain viable after NETosis under certain circumstances. To date, research has focused on the mechanisms underlying NETosis and roles of various factors such as reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH), myeloperoxidase (MPO), and peptidyl arginine deiminase 4 (PAD4). Metabolic pathways such as glycolysis are critical for NET formation, with exogenous glucose and glutamine enhancing NET release. Neutrophils cultured in glucose-free conditions fail to undergo NETosis upon phorbol-12-myristate-13-acetate (PMA) stimulation. ROS-mediated signaling promotes NE release from the azurosome, F-actin degradation, and NE translocation to the nucleus, facilitating chromatin decondensation. Notably, rapid F-actin disassembly has similarly been observed during NETosis induced by PMA and ionomycin. Recently, the role of NSPs during NET formation and their extracellular functions have received increased attention from researchers. The exact mechanism of NET formation remains unknown, and the process itself still raises controversies regarding its overlapping aspects with other forms of cell death, the role of NSPs, the nature of scaffolding DNA, and the possible involvement of other factors. Here, we discuss the intricate pathways governing NET formation, outline the diverse enzymes and proteins crucial for NET assembly, and highlight potential mechanisms controlling NET release. We pay particular attention to the regulation of NSP proteolytic activity and the nuanced role of NSPs during processes such as degranulation, which can be classified as extracellular mechanisms associated with NET formation. Dysregulated NETosis and NSP activity have been implicated in pathological states and diseases. Therefore, understanding the functions of NSPs and their role in NET formation might facilitate the development of new diagnostic and therapeutic strategies.

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.

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation; and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.