Deubiquitinating enzyme USP9X regulates cellular clock function by modulating the ubiquitination and degradation of a core circadian protein BMAL1
Deubiquitinating enzyme USP9X regulates cellular clock function by modulating the ubiquitination and degradation of a core circadian protein BMAL1
复制标题
去泛素化酶 USP9X 通过调节核心昼夜节律蛋白 BMAL1 的泛素化和降解来调节细胞时钟功能
DOI：
10.1042/bcj20180005
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发表时间：
2018-04-30
影响因子：
4.1
通讯作者：
Xu, Guoqiang
中科院分区：
文献类型：
--
作者：
Zhang, Yang;Duan, Chunyan;Xu, Guoqiang
地球上的生物体维持着大约24小时的昼夜节律，这是由生物钟基因及其蛋白质产物调节的。核心时钟蛋白的翻译后修饰可以影响昼夜节律行为。虽然核心时钟蛋白的泛素化被广泛研究，但逆过程去泛素化才刚刚开始展开，这种调节对昼夜节律功能的作用还没有完全了解。在这里，我们使用亲和纯化和质谱分析，以确定可能的泛素羧基末端水解酶FAF-X（USP 9 X）的核心时钟蛋白芳烃受体核转位样蛋白1（ARNTL或BMAL 1）的相互作用蛋白。通过生物化学实验，我们发现USP 9 X减少了BMAL 1的泛素化，增强了其稳定性，并增加了其蛋白水平，导致转录活性升高。生物发光测量表明，USP 9 X敲低降低了细胞昼夜节律的幅度，但周期和相位不受影响。我们的实验在翻译后水平上发现了一种新的生物钟调节因子，并通过转录-翻译反馈环正向分支的核心时钟蛋白BMAL 1的去泛素化和上调来证明生物钟的不同调节功能。
Living organisms on the earth maintain a roughly 24 h circadian rhythm, which is regulated by circadian clock genes and their protein products. Post-translational modifications of core clock proteins could affect the circadian behavior. Although ubiquitination of core clock proteins was studied extensively, the reverse process, deubiquitination, has only begun to unfold and the role of this regulation on circadian function is not completely understood. Here, we use affinity purification and mass spectrometry analysis to identify probable ubiquitin carboxyl-terminal hydrolase FAF-X (USP9X) as an interacting protein of the core clock protein aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL or BMAL1). Through biochemical experiments, we discover that USP9X reduces BMAL1 ubiquitination, enhances its stability, and increases its protein level, leading to the elevated transcriptional activity. Bioluminescence measurement reveals that USP9X knockdown decreases the amplitude of the cellular circadian rhythm but the period and phase are not affected. Our experiments find a new regulator for circadian clock at the post-translational level and demonstrate a different regulatory function for the circadian clock through the deubiquitination and the up-regulation of the core clock protein BMAL1 in the positive limb of the transcription-translation feedback loop.