Protein:Protein Interaction Networks in the Circadian Clock

描述(申请人提供)：在这里，我们使用一种创新的、结合化学和生物物理的方法来破译生物钟用于将复杂的环境感知途径整合到新陈代谢和发育调节中的分子机制。细胞生理学与环境变量的昼夜变化同步是昼夜节律时钟的中心方面。值得注意的是，由于睡眠周期中断或代谢功能改变而导致的主时钟和外周时钟的不同步与肥胖、糖尿病和心脏病等疾病的发生和发展有关。值得注意的是，脊椎动物体内携带途径的复杂性和数量阻碍了代谢和核心昼夜节律振荡器相互调节的分子机制。相比之下，调节植物昼夜节律功能的主要环境变量是蓝光，这使得对蛋白质的精确时空询问成为可能：蛋白质相互作用网络将环境因素整合到新陈代谢和发育的昼夜调节中。在这里，我们集中于阐明黄素结合的光受体在模式生物拟南芥中调节昼夜节律功能的作用。完全了解黄素化学如何以昼夜节律的方式决定蛋白质降解途径的激活，有助于分析包括人类在内的高等生物体中类似的环境敏感途径。为了描绘从最初的光子吸收到生物体生理变化的反应轨迹，我们将重点关注三个主要因素。1)确定刺参昼夜钟光感受器的化学和光化学激活机制。Zeitlupe(ZTL)、Flavin-Kelch-Fbox-1(FKF1)和LOV-Kelch-Protein(LKP2)家族的光感受器通过调节光激活的F-Box结构域，将黄素结合域的激活与蛋白质稳定性的改变结合在一起。结合光谱技术，我们将揭示调控ZTL/FKF1/LKP2功能的化学机制。2.)阐明调控环境感知的结构动力学。结合使用核磁共振和X射线结晶学，我们将破译原子分辨率细节，了解黄素化学的变化如何指示蛋白质结构的变化，从而选择性地激发多个信号通路。3.)蛋白质的调节：蛋白质网络。分子的结构表征 光激活过程将指导蛋白质变体的设计，以选择性地干扰信号复合体的形成。相互作用表面的映射将有助于细胞信号的询问。

DESCRIPTION (provided by applicant): Here we use an innovative, combined chemical and biophysical approach to decipher the molecular mechanism circadian clocks use to integrate complex environmental sensing pathways into regulation of metabolism and development. Synchronization of cellular physiology with diurnal changes in environmental variables is a central aspect of circadian clocks. Notably, desynchronization of master and peripheral clocks due to disruption in sleep cycles or altered metabolic function have been implicated in the onset and progression of diseases ranging from obesity, diabetes and heart disease. Notably, the molecular mechanisms gating reciprocal regulation of metabolism and the core circadian oscillator have been hampered by the complexity and number of entrainment pathways in vertebrates. In contrast, the principal environmental variable regulating circadian function in plants is blue-light, enabling precise spatial and temporal interrogation of protein:protein interaction networks integrating environmental factors into circadian regulation of metabolism and development. Herein, we focus on elucidating the role of flavin- binding photoreceptors in mediating circadian function in the model organism Arabidopsis thaliana. A complete understanding of how flavin chemistry dictates activation of protein degradation pathways in a circadian manner can facilitate analysis of similar environmentally sensitive pathways in higher organisms including humans. To map a reaction trajectory beginning from initial photon absorption to alteration of organism physiology we will focus on three primary factors. 1.) Define the chemical and photochemical activation mechanisms of A. thaliana circadian clock photoreceptors. The Zeitlupe (ZTL), Flavin-Kelch-Fbox-1 (FKF1) and LOV-Kelch-Protein (LKP2) family of photoreceptors couples activation of a flavin-binding domain to alteration in protein stability though regulation of light-activated F-Box domains. Using a combination of spectroscopic techniques we will unravel chemical mechanisms regulating ZTL/FKF1/LKP2 function. 2.) Elucidate structural dynamics regulating environmental sensing. Using a combination of NMR and X-ray crystallography, we will decipher atomic resolution detail of how alteration in flavin chemistry dictates alterations in protein structure to selectively excite multple signaling pathways. 3.) Regulation of protein:protein networks. Structural characterization of the photoactivation process will guide design of protein variants to selectively disrupt formation of signaling complexes. Mapping of interaction surfaces will facilitate interrogation of cellular signaling.