Exploring the structural basis for 24-hour timekeeping in mammals

描述(申请人提供)：哺乳动物有一个内部分子时钟，可以将生理协调成与外部太阳日一致的节奏，通过对综合生化过程的峰值活动进行计时来提供增强的进化适应性。体内24小时生物钟的丢失会扰乱生理与行为和外部环境的时间协调，从而导致糖尿病、新陈代谢综合征、癌症和过早衰老。长期目标是对分子时钟如何在人类体内产生24小时计时产生更深层次的机械理解，以便利用这种时间规律来开发新的创新策略来治疗广泛的人类疾病。这个建议的目的是确定主要的昼夜节律转录因子Clock：BMal1转录调控的结构基础，它每天控制近15%的基因组的表达，以驱动生理的昼夜节律。尽管它在人类生理学中至关重要，但人们对是什么控制着从活跃时钟到抑制性时钟的时间转换知之甚少：BMal1复合体创造了分子时钟固有的24小时周期。中心假设是Clock和BMAL1转录激活域使用固有的灵活性来调节激活物和抑制物的结合，从而对分子时钟的24小时计时做出贡献。利用核磁共振波谱、定量生物化学和基于细胞的研究，我们将追求两个具体目标：1)BMAL1激活结构域中的动态构象开关如何调节时钟：BMal1活性；2)与时钟激活结构域结合的竞争如何调节时钟：在正常时钟功能和人类癌症中。我们的创新方法集成了从细胞生物学到溶液核磁共振光谱学的各种技术，以生成与生物医学相关的原子级别的时钟功能洞察。这项拟议的研究意义重大，因为它有望提供根本上的新概念进展，解决时钟：BMal1如何工作来产生24小时分子节律和控制全球动态平衡的问题。最终，这些知识有可能为人类生理学的昼夜节律控制的基础和转译研究提供新的战略。

DESCRIPTION (provided by applicant): Mammals have an internal molecular clock that coordinates physiology into rhythms that coincide with the external solar day, providing enhanced evolutionary fitness by timing the peak activity of integrated biochemical processes. Loss of this internal 24-hour circadian clock leads to diabetes, metabolic syndrome, cancer, and premature aging by disrupting the temporal coordination of physiology with our behavior and the external environment. The long-term goal is to develop a deeper mechanistic understanding of how the molecular clock generates 24-hour timing in humans, in order to capitalize on this temporal regulation to develop new and innovative strategies to treat a broad spectrum of human diseases. The objective in this proposal is to identify the structural basis for transcriptional regulation by the primary circadian transcription factor, CLOCK:BMAL1, which controls expression of nearly 15% of the genome on a daily basis to drive circadian rhythms of physiology. Despite its critical importance in human physiology, very little is known about what governs the temporal switch from active to repressive CLOCK:BMAL1 complexes that create the intrinsic 24-hour period of the molecular clock. The central hypothesis is that CLOCK and BMAL1 transcriptional activation domains use intrinsic flexibility to regulate binding of activator and repressors to contribute to 24-hour timing of the molecular clock. Using nuclear magnetic resonance spectroscopy, quantitative biochemistry and cell-based studies, we will pursue two specific aims investigating 1) how a dynamic conformational switch in the BMAL1 activation domain regulates CLOCK:BMAL1 activity and 2) how competition for binding to the CLOCK activation domain regulates CLOCK:BMAL1 in normal clock function and in human cancer. Our innovative approach integrates diverse techniques from cell biology to solution NMR spectroscopy to generate biomedically relevant, atomic-level insight into clock function. The proposed research is significant, because it is expected to provide fundamentally new conceptual advances that address how CLOCK:BMAL1 works to generate 24-hour molecular rhythms and control global homeostasis. Ultimately, such knowledge has the potential to inform new strategies for basic and translational research into circadian control of human physiology.