Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes
Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes
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DOI：
10.1074/jbc.ra120.013204
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发表时间：
2020-07-10
影响因子：
4.8
通讯作者：
Kasperkiewicz, Paulina
中科院分区：
文献类型：
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作者：
Janiszewski, Tomasz;Kolt, Sonia;Kasperkiewicz, Paulina
自然杀伤（NK）细胞是对抗病毒感染和控制几种癌症类型的关键先天免疫效应子。对于它们的免疫功能，人类NK细胞主要依赖于五种不同的细胞毒性蛋白酶，称为颗粒酶（A/B/H/K/M）。颗粒酶B（GrB）启动至少三种不同的细胞死亡途径，但其功能的关键方面仍然没有探索，因为目前缺乏检测其活性的选择性探针。在这项研究中，我们使用了一组非天然氨基酸来完全映射GrB的底物偏好，证明了以前未知的GrB底物偏好。然后，我们使用这些偏好来设计基于底物的抑制剂和基于GrB活化活性的荧光探针。我们表明，我们的GrB探针不显着与半胱天冬酶反应，使它们成为深入分析GrB定位和功能的理想选择。使用我们淬灭的荧光底物，我们观察到GrB内的人YT细胞的细胞毒性颗粒。当作为细胞毒效应子使用时，负载GrB的YT细胞攻击MDA-MB-231靶细胞，并且活性GrB影响其靶细胞杀伤效率。总之，我们已经开发了一套分子工具，用于研究GrB在NK细胞中的功能，并展示了用酶激活的荧光底物对GrB的非侵入性视觉检测。
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.