Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkiye.
Institute of Life Sciences and Biotechnology, Bezmialem Vakif University, Beykoz, Turkiye.
J Biol Chem. 2023 Dec;299(12):105451. doi: 10.1016/j.jbc.2023.105451. Epub 2023 Nov 10.
Cryptochromes (CRYs) are essential components of the circadian clock, playing a pivotal role as transcriptional repressors. Despite their significance, the precise mechanisms underlying CRYs' involvement in the circadian clock remain incompletely understood. In this study, we identified a rare CRY2 variant, p.Ser420Phe, from the 1000 Genomes Project and Ensembl database that is located in the functionally important coiled-coil-like helix (CC-helix) region. Functional characterization of this variant at the cellular level revealed that p.Ser420Phe CRY2 had reduced repression activity on CLOCK:BMAL1-driven transcription due to its reduced affinity to the core clock protein PER2 and defective translocation into the nucleus. Intriguingly, the CRY2 variant exhibited an unexpected resistance to degradation via the canonical proteasomal pathway, primarily due to the loss of interactions with E3 ligases (FBXL3 and FBXL21), which suggests Ser-420 of CRY2 is required for the interaction with E3 ligases. Further studies revealed that wild-type and CRY2 variants are degraded by the lysosomal-mediated degradation pathway, a mechanism not previously associated with CRY2. Surprisingly, our complementation study with Cry1Cry2 double knockout mouse embryonic fibroblast cells indicated that the CRY2 variant caused a 7 h shorter circadian period length in contrast to the observed prolonged period length in CRY2 cell lines. In summary, this study reveals a hitherto unknown degradation pathway for CRY2, shedding new light on the regulation of circadian rhythm period length.
隐花色素（CRYs）是生物钟的重要组成部分，作为转录抑制剂发挥着关键作用。尽管其意义重大，但 CRYs 参与生物钟的精确机制仍不完全清楚。在这项研究中，我们从 1000 基因组计划和 Ensembl 数据库中鉴定出一种罕见的 CRY2 变体 p.Ser420Phe，该变体位于功能重要的卷曲螺旋样螺旋（CC-helix）区域。在细胞水平上对该变体的功能特征进行分析，结果表明 p.Ser420Phe CRY2 对 CLOCK:BMAL1 驱动的转录的抑制活性降低，这是由于其与核心时钟蛋白 PER2 的亲和力降低以及核内转位缺陷所致。有趣的是，该 CRY2 变体表现出对经典蛋白酶体途径降解的意外抗性，主要是由于与 E3 连接酶（FBXL3 和 FBXL21）的相互作用丧失，这表明 CRY2 的丝氨酸 420 残基是与 E3 连接酶相互作用所必需的。进一步的研究表明，野生型和 CRY2 变体通过溶酶体介导的降解途径降解，这是以前与 CRY2 无关的机制。令人惊讶的是，我们用 Cry1Cry2 双敲除小鼠胚胎成纤维细胞进行的互补研究表明，与在 CRY2 细胞系中观察到的延长周期长度相反，CRY2 变体导致昼夜节律周期长度缩短了 7 小时。总之，这项研究揭示了 CRY2 的一种迄今未知的降解途径，为生物钟周期长度的调节提供了新的视角。
