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A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins
Aim: The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemica modulators of degradation. For example, Hunti...
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Published in: | Acta pharmacologica Sinica 2016-09, Vol.37 (10), p.1307-1314 |
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Main Authors: | , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Aim: The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemica modulators of degradation. For example, Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of miHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies.
Methods: The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads.
Results: We validated that the signals were within the linear detection range and were HI-r-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates, The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers.
Conclusion: We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTr degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies. |
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ISSN: | 1671-4083 1745-7254 |
DOI: | 10.1038/aps.2016.31 |