Abstract
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.
Highlights
In the last two decades, significant advances in the understanding of natural killer (NK) cells have been made (1)
The S1 binding pocket of Granzyme B (GrB) has an unusual preference for aspartic acid (10, 15); we incorporated Asp at P1, and we elongated the peptide up to six amino acids
Granzyme B, a serine protease involved in programmed cell death through the cleavage of caspase substrates (BID and ICAM), is one of the key factors within NK cells and cytotoxic T lymphocytes (CTLs) leading to cell death (10, 12, 15)
Summary
In the last two decades, significant advances in the understanding of natural killer (NK) cells have been made (1) These cytotoxic lymphocytes are key effectors of innate immunity and are involved in viral infection responses as well as controlling several types of tumors. The activation of these cells is initiated by major compatibility complex (MHC) class I protein loss in compromised cells (1). Grs are the family of homologous serine proteases and the five different human granzymes (A/B/H/K/M); the most studied and abundant are granzymes A and B (3, 4) These enzymes are stored within the cytotoxic granules of immune killer cells and detected in primary breast carcinoma and in chondrocytes of articular cartilage (5).
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