Antibody-free detection of cellular neddylation dynamics of Cullin1
| Autor: | Trish T. Hoang, Xiansi Zhao, Michael E. Bembenek, Keith V. Wood, Zhong Hua Yan, Xiaofeng Yang, Marie K. Schwinn, Ping Li, Jingya Ma, William D. Mallender |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
NEDD8 Protein Chemistry HEK 293 cells Biophysics Context (language use) Cell Biology Transfection Subcellular localization Cullin Proteins HCT116 Cells Biochemistry NEDD8 Cell biology 03 medical and health sciences 030104 developmental biology HEK293 Cells Cell culture Humans CUL1 Biological Assay Neddylation Molecular Biology Protein Processing Post-Translational |
| Zdroj: | Analytical biochemistry. 555 |
| ISSN: | 1096-0309 |
| Popis: | Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time. |
| Databáze: | OpenAIRE |
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