| Autor: |
Guerra M; Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL) and University of Heidelberg, 69117 Heidelberg, Germany.; Faculty of Biosciences, Collaboration for Joint Ph.D. Degree between EMBL and Heidelberg University, 69117 Heidelberg, Germany.; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 69120 Heidelberg, Germany., Halls VS; Dept. of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, Oregon 97239, United States., Schatterny J; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 69120 Heidelberg, Germany., Hagner M; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 69120 Heidelberg, Germany., Mall MA; Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL) and University of Heidelberg, 69117 Heidelberg, Germany.; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 69120 Heidelberg, Germany.; Dept. of Pediatric Pulmonology, Immunology and Critical Care Medicine, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany.; Berlin Institute of Health, 10178 Berlin, Germany.; German Center for Lung Research (DZL), Associated Partner Site, 13353 Berlin, Germany., Schultz C; Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL) and University of Heidelberg, 69117 Heidelberg, Germany.; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 69120 Heidelberg, Germany.; Dept. of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, Oregon 97239, United States. |
| Abstrakt: |
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. |
