Dissecting the Molecular Regulatory Mechanism of Mammalian Circadian Core Compone

描述(申请人提供)：昼夜节律是一种内在的大约24小时的生物钟，嵌入大多数生物体内。在哺乳动物中，昼夜节律在每日周期中协调睡眠-醒来周期、血压、体温和肝脏新陈代谢。在人类中，长期扰乱昼夜节律会损害身心健康。例如，睡眠障碍与昼夜节律有关，影响约20%的美国人，导致更高的医疗成本和生产力下降。开发针对昼夜节律相关疾病的新的治疗药物需要更好地了解调节昼夜节律的机制。人们普遍认为，细胞内的昼夜节律是由转录-翻译环路控制的。在这个环中，CLOCK(昼夜节律运动输出周期kaput)和ARNTL(芳烃受体核转运体样物)形成一个异源二聚体，与称为E-Box的DNA转录元件结合。Clock/ARNTL/E-Box复合体激活PER(PERIOD)和CRY(隐花色素)等昼夜节律基因的转录。翻译后，PER和CREY下调CLOCK/ARNTL的活动。这产生了一个时间延迟的负反馈循环，它设置了时钟相关基因的节奏表达。在理解昼夜节律的基本生物学方面已经取得了重大进展。然而，由于缺乏对昼夜节律的关键功能成分的结构研究，限制了我们对这一重要发条的分子机制的理解。为了克服这一障碍，将结合结构生化工具、传统生化方法和生物物理方法来研究哺乳动物生物钟中核心调控元件之间的相互作用。这项研究将针对两个领域：(1)在转录-翻译环路中作为积极因素的时钟/ARNTL/E-Box复合体；(2)负反馈因素CRY和PER。此外，该项目还将研究一种重要的代谢辅助因子NAD(P)H对昼夜节律复合体的影响，该复合体将把细胞代谢与昼夜节律联系起来。将表达哺乳动物时钟、ARNTL、PER和CRY，并将利用传统的生化和生物物理实验，如电泳、高效液相、紫外或荧光光谱、动态光散射和表面等离子体共振，在体外组装和分析它们的复合体。单个蛋白质和蛋白质/DNA复合体的结构将通过X射线结晶学、低温电子显微镜和小角X射线散射来确定。然后，这些结构将被用来确定蛋白质/DNA、蛋白质/蛋白质和蛋白质/配体界面上的关键残基，这些残基对Circadia核心成分之间的相互作用至关重要。从所获得的各种结构中可以得出关于昼夜节律调节机制的结论，这将为未来开发针对昼夜节律紊乱的新的治疗策略提供必要的知识。

DESCRIPTION (provided by applicant): Circadian rhythm is an intrinsic roughly-24-hour biological clock embedded within most living organisms. In mammals, circadian rhythm coordinates sleep-wake cycles, blood pressure, body temperature and liver metabolism in a daily cycle. In humans, long term disruption of circadian rhythm can impair physical and mental health. For instance, sleep disorders are circadian related and affect about 20% of Americans, resulting in higher healthcare costs and lost productivity. Developing new therapeutic agents against circadian related diseases requires a better understanding of the mechanism involved in regulating the circadian rhythm. It is widely accepted that the circadian rhythm is governed within the cell by a transcriptional-translational loop. In this loop, CLOCK (circadian locomotor output cycle kaput) and ARNTL (aryl hydrocarbon receptor nuclear translocator-like) form a heterodimer that binds to a DNA transcriptional element termed E-Box. The CLOCK/ARNTL/E-Box complex activates the transcription of circadian genes such as PER (period) and CRY (cryptochrome). After translating, PER and CRY down-regulate CLOCK/ARNTL's activity. This generates a time delayed negative feedback loop that sets the rhythmic expression of clock-related genes. Significant progress has been made towards understanding the basic biology of the circadian rhythm. However, the lack of structural studies of the key functional components of the circadian rhythm limits our understanding of the molecular mechanism of this important clockwork. In order to overcome this barrier, combinations of structural biochemical tools, traditional biochemical, and biophysical methods will be applied to investigate the interactions among core regulatory components in the mammalian circadian clockwork. The research will target two areas: (1) the CLOCK/ARNTL/E-Box complex that serves as the positive factor in the transcriptional- translational loop and (2) the negative feedback factors CRY and PER. In addition, the project will study the effect of an important metabolism cofactor, NAD (P) H, on the circadian complexes, which will link the cellular metabolism to the circadian rhythm. Mammalian CLOCK, ARNTL, PER and CRY will be expressed and their complexes will be in vitro assembled and analyzed using traditional biochemical and biophysical experiments such as electrophoresis, liquid chromatography, UV or fluorescent spectra, dynamic light scattering and surface plasmon resonance. Structures of individual proteins and protein/DNA complexes will be determined by X-ray crystallography, cryo-electron microscopy and small angle X-ray scattering. These structures will then be used to identify the key residues at the protein/DNA, protein/protein, and protein/ligand interface that are critical to the interaction between circadia core components. Conclusions regarding regulatory mechanisms of the circadian rhythm can be drawn from the various structures obtained, which will provide essential knowledge for future development of new therapeutic strategies against circadian disorders.