CLOCKΔ19结构域的突变扫描识别出调节生物钟动力学和共调节剂结合的氨基酸,Acta Physiologica

本期发表的一种新的基于细胞培养的敲除和拯救系统提供了对生物钟蛋白 CLOCK（昼夜运动输出循环 Kaput）特定氨基酸的功能洞察。1通过系统地突变功能重要的 Δ19 结构域中的单个氨基酸，2 Abdo 等人1确定了对 CLOCK/BMAL1 寡聚化和昼夜动力学至关重要的氨基酸，包括对共同调节因子结合很重要的氨基酸。这些结果为更详细的分子理解昼夜节律产生机制提供了基础。

时钟是通过化学诱变筛选提出的，作为哺乳动物昼夜节律钟的第一个基因成分。2, 3化学诱变通过突变 RNA 剪接位点删除了鼠类Clock基因的外显子 19。从这个突变基因产生的蛋白质被称为 CLOCKΔ19。低磷酸化减弱了它的降解。4因此，具有这种突变的小鼠过度表达 CLOCKΔ19 蛋白，这会延长在持续黑暗中以心律失常结束的昼夜节律。CLOCKΔ19 中缺乏的 51 个氨基酸（图 1，Δ19）影响 CLOCK/BMAL1 介导的含有启动子的 E-box 的反式激活，并允许延长的卷曲螺旋二聚体形成。后一种结构是一种反平行亮氨酸拉链（图 1，紫色渐变），它可以与一种称为 CLOCK 相互作用蛋白昼夜节律 (CIPC) 的蛋白质共结晶。5CLOCK 线圈与 CIPC 的相互作用可能通过 CLOCK 磷酸化和降解影响 CLOCK:BMAL1 活性。Δ19结构域可能参与形成存在于几个E-box（例如串联E-box）上的CLOCK：BMAL1超复合物以形成调节蛋白识别的用于激活或抑制的大分子复合物。然而，Δ19 结构域调节 CLOCK:BMAL1 介导的转录的确切作用尚不清楚。

在 Abdo 等人1在本期提出的新工作中，作者系统地将 Δ19 结构域中的每一个氨基酸突变为丙氨酸（48 个残基）或精氨酸（3 个 Ala 残基），并研究了它们对细胞昼夜节律性的影响。在定点诱变后，使用慢病毒系统将 CLOCK 变体转染到缺乏Clock基因的人Bmal1-荧光素酶报告细胞中。细胞被同步并记录了几天的生物发光节律。昼夜周期评估确定了挽救 CLOCKΔ19 表型的点突变。数据是通过野生型 CLOCK 与救援相关的。有趣的是，一些 CLOCK 变体无法挽救Clock的心律失常敲除报告细胞（图 1，以黄色突出显示）。因此，这些是时钟功能的必需氨基酸。其他变体促进了超过 1 小时的更短周期（图 1，以蓝色突出显示）或超过 1 小时的更长周期（图 1，以灰色突出显示），表明 Δ19 域的多种功能。

野生型细胞 ( Clock +/+ ) 中的 CLOCK 变体过表达导致不同的时钟周期效应。那些延长时钟敲除细胞的周期并没有延长野生型细胞的周期，而那些没有挽救时钟敲除细胞的昼夜节律的那些对野生型细胞有不同的影响，再次表明 Δ19 域的多种功能.

在共反式激活分析中，Abdo 等人1测试了 CLOCK 变体激活报告构建体的能力，该构建体包含与荧光素酶报告基因偶联的六个 E 盒。与 BMAL1 共转染揭示了 Δ19 结构域中对反式激活至关重要的几个氨基酸（图 1，带有红色字母的氨基酸）。其他人在这个过程中没有发挥作用（图 1，带有蓝色字母的氨基酸）表明这些氨基酸在负反馈回路中具有其他功能。

总之，Abdo 等人1表明亮氨酸拉链氨基酸对 CLOCK 功能最重要（图 1）。这一观点得到了物种5中这些氨基酸的保守性的支持（图 1，粗体氨基酸）。有趣的是，对于确定周期长度显然很重要的氨基酸位于亮氨酸拉链的 N 或 C 端。因此，这些氨基酸可以调节 CLOCK 蛋白的稳定性。这可能通过激酶和/或共同调节因子（如 CIPC、5 MLL1、6 PASD1 7和其他迄今未确定的因素）的结合而发生。Abdo 等人共享的工具1可以适应于识别 CLOCKΔ19 域的新的功能重要的相互作用伙伴，以解开生物钟机制。

"点击查看英文标题和摘要"

A new cell culture–based knock-out and rescue system published in this issue provides functional insight into specific amino acids of the circadian clock protein CLOCK (Circadian Locomotor Output Cycles Kaput).1 By systematically mutating individual amino acids in the functionally important Δ19 domain,2 Abdo et al1 identify amino acids critical for CLOCK/BMAL1 oligomerization and circadian dynamics, including amino acids important for co-regulator binding. These results provide a basis for a more detailed molecular understanding of the mechanism of circadian rhythm generation.

Clock came up by chemical mutagenesis screening as the first gene component reported of the mammalian circadian clock.2, 3 Chemical mutagenesis deleted exon 19 of the murine Clock gene by mutating an RNA splice site. The resulting protein from this mutated gene is termed CLOCKΔ19. Hypophosphorylization attenuates its degradation.4 Hence, mice with this mutation overexpress CLOCKΔ19 protein, which prolongs the circadian period ending in arhythmicity in constant darkness. The 51 amino acids lacking in CLOCKΔ19 (Figure 1, Δ19) affect CLOCK/BMAL1-mediated transactivation of E-box containing promoters and allow extended coiled-coil dimer formation. The latter structure is an antiparallel leucine zipper (Figure 1, purple gradients) that can co-crystallize with a protein called CLOCK interacting protein circadian (CIPC).5 CLOCK coil interaction with CIPC may influence CLOCK:BMAL1 activity by CLOCK phosphorylation and degradation. The Δ19 domain may be involved in forming CLOCK:BMAL1 super-complexes present on several E-boxes (eg tandem E-boxes) to form a macromolecular complex recognized by regulatory proteins for activation or repression. However, the exact role of the Δ19 domain regulating CLOCK:BMAL1-mediated transcription is not understood.

In the novel work presented in this issue by Abdo et al1 the authors systematically mutate every single amino acid in the Δ19 domain to alanine (48 residues) or to arginine (3 Ala residues) and investigated their influence on cell circadian rhythmicity. After site directed mutagenesis, the CLOCK variants were transfected into human Bmal1-luciferase reporter cells lacking the Clock gene using a lentiviral system. Cells were synchronized and bioluminescence rhythms were recorded for several days. Circadian period assessment identified the point mutation rescuing the CLOCKΔ19 phenotype. The data were set in relation to the rescue by wild-type CLOCK. Interestingly, some of the CLOCK variants were not able to rescue arhythmicity of Clock knock-out reporter cells (Figure 1, highlighted in yellow). Thus, those are the essential amino acids for CLOCK functionality. Other variants promoted a more than 1 hour shorter period (Figure 1, highlighted in blue) or a more than 1 hour longer period (Figure 1, highlighted in grey), suggesting multiple functions of the Δ19 domain.

CLOCK variant overexpression in wild-type cells (Clock+/+) caused diverse clock period effects. Those lengthening the Clock knock-out cells’ period did not lengthen the period in wild-type cells, whereas those not rescuing circadian rhythms of Clock knock-out cells had differential effects on wild-type cells, again indicating multiple functions of the Δ19 domain.

In a co-transactivation assay, Abdo et al1 tested CLOCK variant abilities to activate a reporter construct containing six E-boxes coupled to a luciferase reporter. Co-transfection with BMAL1 revealed several amino acids in the Δ19 domain crucial for transactivation (Figure 1, amino acids with red lettering). Others played no role in this process (Figure 1, amino acids with blue lettering) suggesting other functions for these amino acids in the negative feedback loop.

Taken together, Abdo et al1 show that leucine zipper amino acids are most important for CLOCK function (Figure 1). This view is supported by conservation of these amino acids across species5 (Figure 1, amino acids in bold). Interestingly, apparently important amino acids for period length determination are located at the N- or C-term of the leucine zipper. Accordingly, these amino acids may regulate CLOCK protein stability. This could occur either via binding of kinases and/or co-regulators such as CIPC,5 MLL1,6 PASD17 and other so far unidentified factors. The tools shared by Abdo et al1 can be adapted to identify novel functionally important interacting partners of the CLOCKΔ19 domain to unravel circadian clock machinery.