Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
J Biol Chem. 2023 Mar;299(3):102929. doi: 10.1016/j.jbc.2023.102929. Epub 2023 Jan 20.
Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2, and PER3), CRY (CRY1 and CRY2), and CK1δ. In mice, peak expression of the repressors in the early morning reduces CLOCK- and BMAL1-mediated transcription/translation of the repressors themselves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24 h. Studies have characterized a variety of possible protein interactions and complexes associated with the function of this transcription-translation feedback loop. Our prior investigation suggested there were two circadian complexes responsible for rhythmicity, one containing CLOCK-BMAL and the other containing PER2, CRY1, and CK1δ. In this investigation, we acquired data from glycerol gradient centrifugation and gel filtration chromatography of mouse liver extracts obtained at different circadian times to further characterize circadian complexes. In addition, anti-PER2 and anti-CRY1 immunoprecipitates obtained from the same extracts were analyzed by liquid chromatography-tandem mass spectrometry to identify components of circadian complexes. Our results confirm the presence of discrete CLOCK-BMAL1 and PER-CRY-CK1δ complexes at the different circadian time points, provide masses of 255 and 707 kDa, respectively, for these complexes, and indicate that these complexes are composed principally of the core circadian proteins.
昼夜节律由一组核心时钟蛋白维持,包括转录激活因子 CLOCK 和 BMAL1,以及抑制因子 PER(PER1、PER2 和 PER3)、CRY(CRY1 和 CRY2)和 CK1δ。在小鼠中,抑制因子在清晨的峰值表达降低了 CLOCK 和 BMAL1 介导的抑制因子自身的转录/翻译。到下午晚些时候,抑制因子大部分被降解耗尽,从而在每 24 小时重复的循环中重新激活其表达。研究已经描述了与这种转录-翻译反馈环功能相关的多种可能的蛋白质相互作用和复合物。我们之前的研究表明,有两种昼夜节律复合物负责节律性,一种包含 CLOCK-BMAL,另一种包含 PER2、CRY1 和 CK1δ。在这项研究中,我们从不同昼夜时间点获得的小鼠肝提取物的甘油梯度离心和凝胶过滤层析获得数据,以进一步表征昼夜节律复合物。此外,还对来自相同提取物的抗 PER2 和抗 CRY1 免疫沉淀物进行了液相色谱-串联质谱分析,以鉴定昼夜节律复合物的成分。我们的结果证实了在不同的昼夜时间点存在离散的 CLOCK-BMAL1 和 PER-CRY-CK1δ 复合物,分别为这些复合物提供了 255 和 707 kDa 的分子量,并表明这些复合物主要由核心昼夜节律蛋白组成。