Stamatakou Eleanna, Zhu Ye, Rubinsztein David C
Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2018;1780:17-29. doi: 10.1007/978-1-4939-7825-0_2.
The accumulation of mutant aggregate-prone proteins is a hallmark of the majority of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Autophagy, a cytosolic bulk degradation system, is the major clearance pathway for several aggregate-prone proteins, such as mutant huntingtin. The autophagosome-associated protein LC3-II is a specific marker of autophagic flux within cells, whereas aggregate formation of mutant huntingtin represents a good readout for studying autophagy modulation. Here we describe the method of assessing autophagic flux using LC3-II western blotting and substrate clearance by expressing the N-terminal fragment of huntingtin (htt exon 1) containing an expanded polyglutamine tract in mammalian cells.
突变的易聚集蛋白的积累是大多数神经退行性疾病的标志，包括阿尔茨海默病、帕金森病和亨廷顿舞蹈症。自噬是一种胞质大量降解系统，是几种易聚集蛋白（如突变型亨廷顿蛋白）的主要清除途径。自噬体相关蛋白LC3-II是细胞内自噬流的特异性标志物，而突变型亨廷顿蛋白的聚集形成是研究自噬调节的良好指标。在这里，我们描述了一种通过LC3-II蛋白质印迹法评估自噬流以及通过在哺乳动物细胞中表达含有扩展聚谷氨酰胺序列的亨廷顿蛋白N端片段（htt外显子1）来评估底物清除的方法。
