Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells
| Autor: | David L. Roman, Michael P. Hayes, Christopher Bodle, Joseph B. O’Brien |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
G protein High-throughput screening Protein combining Biophysics GTP-Binding Protein alpha Subunits Gi-Go Biochemistry Article Cell Line RGS4 03 medical and health sciences Regulator of G protein signaling Protein-fragment complementation assay Animals Humans Luciferase Molecular Biology 030102 biochemistry & molecular biology biology fungi Cell Biology Molecular biology Small molecule Rats Cell biology 030104 developmental biology Luminescent Measurements biology.protein GTP-Binding Protein alpha Subunits Gq-G11 Biological Assay RGS Proteins |
| Zdroj: | Analytical Biochemistry. 522:10-17 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2017.01.013 |
| Popis: | Cell based assessment tools and screening platforms are the preferred paradigm for small molecule identification and validation due to selectively identifying molecules with cellular activity and validation of compound activity against target proteins in their native environment. With respect to Regulator of G Protein Signaling (RGS) proteins, current cell based methodologies are either low throughput or monitor downstream signaling consequences. The increasing number of reports indicating RGS function in various disease pathogeneses highlights the need for a robust RGS inhibitor discovery and characterization paradigm. Promega’s NanoBit Protein Complementation Assay utilizes NanoLuc, an engineered luciferase with enhanced luminescence characteristics which allow for both robust and kinetic assessment of protein interaction formation and disruption. Here we characterized 15 separate RGS: G protein interactions using this system. The binding profile of RGS: Gα interactions correlates to prior published biochemical binding profiles of these proteins. Additionally, we demonstrated this system is suitable for high throughput screening efforts via calculation of Z-factors for three of the interactions and demonstration that a known small molecule inhibitor of RGS4 disrupts the RGS4: Gαi1 protein-protein interaction. In conclusion, the NanoBit Protein Complementation Assay holds promise as a robust platform for discovery and characterization of RGS inhibitors. |
| Databáze: | OpenAIRE |
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