A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins.
OBJECTIVE
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 chemical 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 mHTT 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 HTT-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.


CONCLUSIONS
We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT 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.
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
Peng
P
Wu
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
Ming-Xing
MX
Lu
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
Xiao-Tian
XT
Cui
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
He-Qing
HQ
Yang
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
Shen-Liang
SL
Yu
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
Jian-Bin
JB
Zhu
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
Xiao-Li
XL
Sun
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Brain Science, School of Life Sciences, Fudan University, Shanghai 200433, China.
Boxun
B
Lu
D000818:Animals; D002460:Cell Line; D057930:Click Chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D000071058:Huntingtin Protein / Q000378:metabolism*; D051379:Mice; D059748:Proteolysis
D000071058:Huntingtin Protein