Investigation of osteoclast cathepsin K activity in osteoclastogenesis and bone loss using a set of chemical reagents.

Cathepsin K (CatK) is a lysosomal cysteine protease whose highest expression is found in osteoclasts, which are the cells responsible for bone resorption. Investigations of the functions and physiological relevance of CatK have often relied on antibody-related techniques, which makes studying its activity patterns a challenging task. Hence, we developed a set of chemical tools for the investigation of CatK activity. We show that our probe is a valuable tool for monitoring the proteolytic activation of CatK during osteoclast formation. Moreover, we demonstrate that our inhibitor of CatK impedes osteoclastogenesis and bone resorption and that CatK is stored in its active form in osteoclasts within their lysosomal compartment and mainly in the ruffled borders of osteoclasts. Given that our probe recognizes active CatK within living cells without exhibiting any observed cytotoxicity in the several models tested, we expect that it would be well suited to theranostic applications in CatK-related diseases.

Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes.

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.

Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe.

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.

NETosis occurs independently of neutrophil serine proteases.

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.

Determination of extended substrate specificity of the MALT1 as a strategy for the design of potent substrates and activity-based probes.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) belongs to the CD clan of cysteine proteases. MALT1 is a unique enzyme among this clan because it recognizes the basic amino acid arginine in the P1 pocket. Previous studies carried out with natural amino acids revealed the substrate specificity of the P4-P1 pockets of MALT1 but have provided only limited information about the catalytic preferences of this enzyme. In this study, we exploited Hybrid Combinatorial Substrate Library and Internally Quenched Fluorescence substrate technologies to interrogate the extended substrate specificity profile of the S5-S2' active site pockets using unnatural amino acids. This strategy resulted in the design of a peptide-based fluorogenic substrate, which exhibited significant activity toward MALT1. Subsequently, the substrate sequence was further utilized to develop potent, irreversible activity-based probes.