Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux.
Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux.
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DOI：
10.3791/60133
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发表时间：
2019-09
期刊：
影响因子：
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通讯作者：
Mikhail Ryzhikov;Anna Eubanks;J. Haspel
中科院分区：
文献类型：
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作者：
Mikhail Ryzhikov;Anna Eubanks;J. Haspel
细胞采用几种方法来回收不需要的蛋白质和其他物质，包括溶酶体和非溶酶体途径。主要的溶酶体依赖性途径称为自噬，而蛋白质催化的主要非溶酶体方法是泛素-蛋白酶体系统。最近在模式生物中的研究表明，自噬和泛素-蛋白酶体系统的活性在一天中并不恒定，而是根据每日（昼夜）节律而变化。测量蛋白质周转中的生物节律的能力对于理解如何实现细胞质量控制和理解感兴趣的特定蛋白质的动力学是重要的。在这里，我们提出了一个标准化的协议，用于量化体内自噬和蛋白酶体通量，捕获蛋白质周转的昼夜组成部分。我们的方案包括使用小鼠肝脏作为起始材料的小鼠处理、组织处理、分级分离和自噬通量定量的细节。
Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.