A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins  

作　　者：Peng WU Ming-xing LU Xiao-tian CUI He-qing YANG Shen-liang YU Jian-bin ZHU Xiao-li SUN Boxun LU 

机构地区：[1]State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, FudanUniversity, Shanghai 200433, China

出　　处：《Acta Pharmacologica Sinica》2016年第10期1307-1314,共8页中国药理学报（英文版）

基　　金：Acknowledgements The study was supported by the National High Technology Research and Development Program of China （2014AA02502） and the National Natural Science Foundation of China （NSFC 31371421 and 31422024）.

摘　　要：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.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.

关 键 词：click chemistry SPAAC HIGH-THROUGHPUT PULSE-CHASE protein degradation Huntington＇s disease PolyQ 

分 类 号：R[医药卫生]