Peng Jiawen, Liu Na, Ren Yixuan, Wang Jiahui, Jin Yanxia, Wang Xianping, Wang Weidong, Pan Jicheng
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, College of Life Sciences, Hubei Normal University, Huangshi 435002, China.
Int J Mol Sci. 2025 Aug 18;26(16):7962. doi: 10.3390/ijms26167962.
Mammalian circadian rhythms, governing ~24 h oscillations in behavior, physiology, and hormone levels, are orchestrated by transcriptional-translational feedback loops centered around the core clock protein cryptochrome 1 (CRY1). While CRY1 ubiquitination is known to regulate clock function, the roles of specific ubiquitination sites remain unclear. Here, we identify lysine 151 (K151) as a critical residue modulating the circadian period through non-canonical mechanisms. Using site-directed mutagenesis, we generated CRY1-K151Q/R mutants mimicking constitutive deubiquitination. Circadian rescue assays in Cry1/2-deficient cells revealed period shortening (K151Q: -2.25 h; K151R: -1.4 h; n = 3, < 0.01, Student's -test), demonstrating K151's functional importance. Despite normal nuclear localization kinetics, K151Q/R mutants exhibited reduced transcriptional repression in luciferase assays, a weakened interaction with BMAL1 by the luciferase complementation assay, and enhanced binding to E3 ligase FBXL12 (but not FBXL3) while showing more stability than wild-type CRY1. Notably, the absence of ubiquitination-linked degradation or altered FBXL3 engagement suggests a ubiquitination-independent mechanism. We propose that CRY1-K151 serves as a structural hub fine-tuning circadian periodicity by modulating core clock protein interactions rather than through traditional ubiquitin-mediated turnover. These findings redefine the mechanistic landscape of post-translational clock regulation and offer new therapeutic avenues for circadian disorders.
哺乳动物的昼夜节律控制着行为、生理和激素水平约24小时的振荡，由围绕核心时钟蛋白隐花色素1（CRY1）的转录-翻译反馈环精心编排。虽然已知CRY1泛素化调节时钟功能，但特定泛素化位点的作用仍不清楚。在这里，我们确定赖氨酸151（K151）是一个关键残基，通过非经典机制调节昼夜节律周期。使用定点诱变，我们生成了模拟组成型去泛素化的CRY1-K151Q/R突变体。在Cry1/2缺陷细胞中的昼夜节律拯救试验显示周期缩短（K151Q：-2.25小时；K151R：-1.4小时；n = 3，P < 0.01，学生t检验），证明了K151的功能重要性。尽管核定位动力学正常，但K151Q/R突变体在荧光素酶测定中表现出转录抑制降低，在荧光素酶互补测定中与BMAL1的相互作用减弱，与E3连接酶FBXL12（而非FBXL3）的结合增强，同时比野生型CRY1更稳定。值得注意的是，缺乏泛素化相关降解或改变的FBXL3参与表明存在一种不依赖泛素化的机制。我们提出CRY1-K151作为一个结构枢纽，通过调节核心时钟蛋白相互作用而非传统的泛素介导的周转来微调昼夜节律周期。这些发现重新定义了翻译后时钟调节的机制格局，并为昼夜节律紊乱提供了新的治疗途径。
