Protease FRET Reporters Targeting Neutrophil Extracellular Traps,Journal of the American Chemical Society

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Protease FRET Reporters Targeting Neutrophil Extracellular Traps
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2020-11-13 , DOI: 10.1021/jacs.0c08130
Matteo Guerra _{1,

2,

3} , Victoria S Halls ₄ , Jolanthe Schatterny ₃ , Matthias Hagner ₃ , Marcus A Mall _{1,

3,

5,

6,

7} , Carsten Schultz _{1,

3,

4}

Affiliation

Neutrophil extracellular traps (NETs) consist of DNA released by terminally stimulated neutrophils. They fine-tune inflammation, kill pathogens, activate macrophages, contribute to airway mucus obstruction in cystic fibrosis, and facilitate tumor metastasis after dormancy. Neutrophil proteases such as elastase (NE) and cathepsin G (CG) attach to NETs and contribute to the diverse immune outcome. However, because of the lack of suitable tools, little spatiotemporal information on protease activities on NETs is available in a pathophysiological context to date. Here, we present H-NE and H-CG, two FRET-based reporters armed with a DNA minor groove binder, which monitor DNA-bound NE and CG activity, respectively. The probes revealed that only NE maintains its catalytic ability when localized to DNA. Further, we demonstrated elevated protease activity within the extracellular DNA of sputum from cystic fibrosis patients. Finally, H-NE showed NE activity at single-cell and free DNA resolution within mouse lung slices, a difficult to achieve task with available substrate-based reporters.

中文翻译：

靶向中性粒细胞胞外陷阱的蛋白酶 FRET 报告基因

中性粒细胞胞外陷阱 (NET) 由最终刺激的中性粒细胞释放的 DNA 组成。它们微调炎症，杀死病原体，激活巨噬细胞，导致囊性纤维化中的气道粘液阻塞，并促进休眠后的肿瘤转移。中性粒细胞蛋白酶，如弹性蛋白酶 (NE) 和组织蛋白酶 G (CG) 附着在 NET 上，有助于产生不同的免疫结果。然而，由于缺乏合适的工具，迄今为止，在病理生理学背景下，关于 NET 上蛋白酶活性的时空信息很少。在这里，我们提出了 H-NE 和 H-CG，这是两种基于 FRET 的报告基因，配备有 DNA 小沟结合物，分别监测 DNA 结合的 NE 和 CG 活性。探针显示，只有 NE 在定位于 DNA 时仍保持其催化能力。此外，我们还证明了囊性纤维化患者痰细胞外 DNA 中的蛋白酶活性升高。最后，H-NE 在小鼠肺切片中显示出单细胞和游离 DNA 分辨率的 NE 活性，这是利用现有的基于底物的报告基因很难完成的任务。

更新日期：2020-11-13

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