The Linchpin that Joins the Circadian Oscillator to Clock Output

描述（由申请人提供）：生物体通过系统地改变其代谢、生理和行为，与昼夜同步，利用可预测的环境光/暗周期。这些由分子钟产生的昼夜节律如果被打乱，会对健康和疾病产生深远的影响。然而，这些生物钟的机制在任何生物体中都只被部分理解。由于对这些机制的严格理解对于解决与昼夜节律相关的疾病是必不可少的，因此LiWang研究小组的长期目标是阐明模型系统中时钟和时钟控制细胞过程的机制。该实验室的文献报告和数据强烈表明，时钟蛋白重新排列其整体构象，从而重新排列其功能特性，作为计时机制的一部分。例如，领跑者蛋白，动物中的PER和真菌中的FRQ，随着时间的推移，会在全局紧凑状态和开放状态之间发生构象变化。同样，我们最近发现蓝藻时钟蛋白KaiB，也会进行全局构象重排，KaiB ？KaiB*，称为“折叠开关”。令人惊讶的是，我们还发现KaiB折叠开关不仅在昼夜节律的产生中起着至关重要的作用，而且还调节这些节律的下游传递。因此，本文的目的是阐明蛋白质在时钟机制中大规模构象变化的作用。预计实现这一目标将对原核生物和真核生物的时间生物学领域产生巨大影响。这个提议的中心假设是KaiB ？KaiB*折叠开关是连接振荡器功能和时钟输出的关键。为了验证中心假设，我们将追求三个具体目标：1)建立KaiB ？振荡器函数中的KaiB*折叠开关；2)如何确定KaiB ？KaiB*折叠开关调节SasA输出通路；3)确定KaiB ？KaiB*折叠开关调节CikA输出通路。采用综合方法获得的初步数据有力地支持了中心假设：结构？突变体”?体外实验？计算建模？体内实验。在每个领域拥有专业知识的强大合作者团队可以提高所提议工作的可行性。这一建议是创新的，因为缺乏文献报道表明，这一建议的核心概念被忽视了：蛋白质结构的巨大变化支撑着生物钟的机制。这一建议意义重大，因为这里的发现有望为真核生物时钟以及蛋白质折叠开关可能以前未被认识到的其他过程提供新的和可操作的见解。最终，这些知识有可能创造出应对昼夜节律相关疾病的策略。

DESCRIPTION (provided by applicant): Organisms exploit predictable environmental light/dark cycles by systematically varying their metabolism, physiology, and behavior in synchrony with day and night. These circadian rhythms, which are produced by molecular clocks, can have profound consequences to health and disease if disrupted. However, the mechanisms of these circadian clocks are only partially understood in any organism. Because a rigorous understanding of these mechanisms will be indispensable for tackling circadian elated diseases, the long-term goal of the LiWang research group is to elucidate the mechanisms of clocks and clock control over cellular processes in model systems. Reports in the literature and data from this laboratory strongly suggest that clock proteins rearrange their global conformations, and thus their functional properties, as part of the timekeeping mechanism. For example, pace-setter proteins, PER in animals and FRQ in fungi, undergo conformational changes between globally compact and open states as they keep time. Similarly, we recently discovered that the cyanobacteria clock protein, KaiB, also executes global conformational rearrangements, KaiB ? KaiB*, called "fold switching". Surprisingly, we also found that KaiB fold switching not only plays an essential role in the generation of circadian rhythms, but regulates the transmission of those rhythms downstream. Thus, the objective here is to elucidate the roles of large-scale conformational changes by proteins in clock mechanisms. Attaining this goal is predicted to have an enormous influence on the field of both prokaryotic and eukaryotic chronobiology. The central hypothesis of this proposal is that KaiB ? KaiB* fold switching is the linchpin that joins oscillator function to clock output. To test the central hypothesis we will pursue three specific aims: 1) Establish the role of KaiB ? KaiB* fold switching in oscillator functions; 2) Determine how KaiB ? KaiB* fold switching regulates the SasA output pathway; and 3) Determine how KaiB ? KaiB* fold switching regulates the CikA output pathway. The central hypothesis is strongly supported by preliminary data obtained by using an integrative approach: structures ? mutants' ? in vitro experiments ? computational modeling ? in vivo experiments. A strong team of collaborators with expertise in each area enhances the feasibility of the work proposed. This proposal is innovative, because a lack of reports in the literature reveals that the central concept of this proposal has been overlooked: Large changes in protein structure underpin the mechanisms of circadian clocks. The proposal is significant, because the findings here are expected to open new and actionable insights into eukaryotic clocks, and to other processes in which protein fold switching may not have been previously recognized. Ultimately, such knowledge has the potential to create strategies with which to tackle circadian-related diseases.