Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation
Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation
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DOI：
10.1073/pnas.2318274120
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发表时间：
2023-12-26
影响因子：
11.1
通讯作者：
Liu，Yi
中科院分区：
文献类型：
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作者：
Xie，Pancheng;Xie，Xiaowen;Liu，Yi
液-液相分离(LLP)是多种生物过程的基础。由于大多数LLP研究是在体外使用重组蛋白或在过度表达蛋白质的细胞中进行的，LLP与内源性蛋白的生理相关性往往不清楚。Period是一种内在无序的富含结构域的蛋白质，是哺乳动物昼夜节律的中心时钟成分，并在核心昼夜节律负反馈环中与其他时钟蛋白相互作用。不同的核心时钟蛋白以前被证明形成大的复合体。生物钟研究通常依赖于过度表达时钟蛋白的实验。在这里，我们证明了当PER2转基因在细胞中稳定表达时，PER2蛋白形成了依赖于核磷酸化的缓慢移动的LLP凝聚体，该凝聚体招募了其他时钟蛋白。然而，内源PER2的超分辨显微镜显示，它们形成了昼夜节律控制的、快速扩散的核微体，这些微体抵抗蛋白质浓度变化、己二醇处理和磷酸化丧失，表明它们不同于由蛋白质过度表达引起的LLP凝聚体。令人惊讶的是，只有一小部分内源性PER2微体与内源性BMAL1和CRY1瞬时相互作用，这一结论在细胞和小鼠组织中得到了证实，表明在昼夜负反馈过程中存在类似酶的机制。综上所述，这些结果表明，核心时钟蛋白的动态相互作用是哺乳动物生物钟机制的一个关键特征，以及在LLP和生物钟研究中检查内源蛋白的重要性。
Liquid–liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.