Dissecting the Molecular Regulatory Mechanism of Mammalian Circadian Core Compone

描述（由申请人提供）：昼夜节律是嵌入在大多数生物体内的内在的大约24小时的生物钟。在哺乳动物中，昼夜节律在日常循环中协调睡眠-觉醒周期、血压、体温和肝脏代谢。在人类中，昼夜节律的长期破坏会损害身心健康。例如，睡眠障碍与昼夜节律有关，影响了大约20%的美国人，导致更高的医疗成本和生产力损失。开发针对昼夜节律相关疾病的新治疗剂需要更好地了解昼夜节律调节的机制。人们普遍认为，昼夜节律是由细胞内的转录-翻译循环控制的。在这个循环中，CLOCK（昼夜运动输出周期终止）和ARNTL（芳烃受体核易位样）形成异源二聚体，与称为E-Box的DNA转录元件结合。CLOCK/ARNTL/E-Box复合体激活昼夜节律基因的转录，如PER（周期）和CRY（隐花色素）。翻译后，PER和CRY下调CLOCK/ARNTL的活性。这就产生了一个时间延迟的负反馈循环，设定了生物钟相关基因的节律性表达。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，缺乏对昼夜节律关键功能成分的结构研究限制了我们对这一重要生物钟的分子机制的理解。为了克服这一障碍，将结合结构生化工具、传统生化和生物物理方法来研究哺乳动物生物钟中核心调控成分之间的相互作用。研究将针对两个方面：(1)CLOCK/ARNTL/E-Box复合物作为转录-翻译环的正因子；(2)CRY和PER的负反馈因子。此外，该项目将研究一种重要的代谢辅助因子NAD (P) H对昼夜节律复合物的影响，该复合物将细胞代谢与昼夜节律联系起来。哺乳动物CLOCK， ARNTL， PER和CRY将被表达，它们的复合物将被体外组装和分析，使用传统的生化和生物物理实验，如电泳，液相色谱，紫外或荧光光谱，动态光散射和表面等离子体共振。单个蛋白质和蛋白质/DNA复合物的结构将通过x射线晶体学、低温电子显微镜和小角度x射线散射来确定。然后，这些结构将用于鉴定蛋白质/DNA、蛋白质/蛋白质和蛋白质/配体界面上的关键残基，这些残基对昼夜节律核心组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，这将为未来针对昼夜节律紊乱的新治疗策略的开发提供必要的知识。

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.