期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 28, 页码 9567-9582出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA120.013204
关键词
amino acid; granzyme B; serine protease; natural killer cells (NK cells); activity-based probe; innate immune system; quenched fluorescence assay; substrate specificity; peptide; protease
资金
- HOMING Programme, a Grant Project of the Foundation for Polish Science - European Union [2016-3/24]
- Wroclaw University of Science and Technology [8201003902]
- L'Oreal Poland scholarship
- Polish Ministry of Science and Higher Education scholarship
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.
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