A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins.
A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins.
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一种高通量兼容测定法，用于测量内源亨廷顿蛋白的降解
DOI：
10.1038/aps.2016.31
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发表时间：
2016-09
影响因子：
8.2
通讯作者：
Lu B
中科院分区：
文献类型：
--
作者：
Wu P;Lu MX;Cui XT;Yang HQ;Yu SL;Zhu JB;Sun XL;Lu B
致病蛋白的积累是许多神经退行性疾病的共同标志。使用高通量相容测定法测量这些蛋白质的降解是鉴定降解的遗传和化学调节剂的高度需要。例如，亨廷顿氏病（HD）是一种无法治愈的遗传性神经退行性疾病，由突变亨廷顿蛋白（mHTT）的细胞毒性引起。mHTT降解的高通量测量在HD药物的发现和研究中具有重要意义。现有的用于此类目的的方法由于依赖于蛋白质标签或泛蛋白合成抑制剂而具有局限性。在这里，我们报告了一种基于Click化学和均匀时间分辨荧光（htf）技术的高通量兼容脉冲追踪方法（CH-chase），用于测量内源性无标签亨廷顿蛋白（HTT）降解。将脉冲标记的蛋白与生物素结合，采用click反应菌株促进的炔叠氮环加成法（SPAAC），并通过测量链霉亲和素珠拉下后HTT htf信号的还原百分比计算追逐信号。我们验证了信号在线性检测范围内，并且是特定于http的。我们成功地测量了内源性HTT的降解在高通量兼容格式使用96孔板。观察了已知改性剂对HTT降解的预测变化，证实了该方法适用于HTT降解改性剂的鉴定。我们已经建立了第一个高通量兼容的检测方法，能够测量内源性，无标签的HTT降解，为HD研究和药物发现提供了有价值的工具。该方法可应用于其他蛋白质，并可促进其他神经退行性疾病和蛋白质病变的研究。
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. 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. 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. 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.