An optical and noninvasive method to detect the accumulation of ubiquitin chains
An optical and noninvasive method to detect the accumulation of ubiquitin chains
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一种检测泛素链积累的光学非侵入性方法
DOI：
10.1002/cbin.11186
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发表时间：
2019
影响因子：
3.9
通讯作者：
Takahiro Sakai
中科院分区：
文献类型：
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作者：
Jun Imai ;Yuuta Koganezawa;Haruka Tuzuki;Ikumi Ishikawa;Takahiro Sakai
过量的多聚泛素化蛋白的积累具有细胞毒性，并且经常在神经退行性疾病患者的病理组织中观察到。因此，利用光学和非侵入性方法检测活细胞中多聚泛素化蛋白数量的增加是一种很有前途的策略，可以找出神经退行性疾病的症状和环境原因，也可以用于识别可以抑制多聚泛素化蛋白聚集的化合物。因此，我们生成了一对在 N 末端标记泛素的荧光蛋白 [Azamigreen (Azg) 和 Kusabiraorange (Kuo)]（Azg-Ub 和 Kuo-Ub），并开发了基于 Azg/Kuo 的荧光共振能量转移 (FRET) 测定法来估计体外和体内多聚泛素链的量。当存在泛素激活酶抑制剂 PYR-41 时，FRET 强度减弱，表明两种荧光泛素与正常泛素一样被纳入泛素链中。添加蛋白酶体抑制剂 MG-132 会增强 FRET 强度，而在自噬激活剂雷帕霉素存在下会降低 FRET 强度，表明带有荧光泛素的泛素链与正常泛素链一样充当降解信号。总之，上述光学方法提供了强大的研究工具来估计体外和体内多聚泛素链的数量，特别是在活细胞中非侵入性地估计。
The accumulations of excess amounts of polyubiquitinated proteins are cytotoxic and frequently observed in pathologic tissue from patients of neurodegenerative diseases. Therefore, optical and non‐invasive methods to detect the increase of the amounts of polyubiquitinated proteins in living cells is a promising strategy to find out symptoms and environmental cause of neurodegenerative diseases, also for identifying compounds that could inhibit gathering of polyubiquitinated proteins. Therefore, we generated a pair of fluorescent protein [Azamigreen (Azg) andKusabiraorange (Kuo)] tagged ubiquitin on its N‐terminus (Azg‐Ub and Kuo‐Ub) and developed an Azg/Kuo‐basedFluorescenceResonanceEnergyTransfer (FRET) assay to estimate the amount of polyubiquitin chains in vitro and in vivo. The FRET intensity was attenuated in the presence of ubiquitin‐activating enzyme inhibitor, PYR‐41, indicating that both fluorescent ubiquitin is incorporated into ubiquitin chains likewise normal ubiquitin. The FRET intensity was enhanced by the addition of the proteasome inhibitor, MG‐132, and was reduced in the presence of the autophagy activator Rapamycin, designating that ubiquitin chains with fluorescent ubiquitin act as the degradation signal equally with normal ubiquitin chains. In summary, the above optical methods provide powerful research tools to estimate the amounts of polyubiquitin chains in vitro and in vivo, especially non‐invasively in living cells.