Molecular interactions of mammalian circadian clock proteins

描述（由申请人提供）：昼夜节律是所有真核生物和一些原核生物的基本特性，允许生物体使其细胞代谢和生理适应地球的~24小时光-暗周期。在哺乳动物中，昼夜节律是由自动调节转录-翻译反馈环产生的，其核心成分是CLOCK、BMAL 1、隐花色素（CRY）和周期（PER）蛋白。异二聚体CLOCK：BMAL 1转录因子激活基因Cry和Per的转录，并反过来被其蛋白产物CRY和PER抑制，最终产生有节奏的基因表达模式。据估计，生物钟影响人类基因组中至少10%的基因的表达，从而影响代谢、生理和行为的许多方面。目前，关于CLOCK：BMAL 1与其负调控因子CRY和PER在生物化学和结构水平上的分子相互作用以及这种相互作用如何调节CLOCK：BMAL 1的活性知之甚少。拟议的研究的目标是阐明CLOCK：BMAL 1，CRY和PER之间的分子相互作用，通过严格的体外生物化学，生物物理学和结构研究，并描绘CLOCK：BMAL 1的转录功能由PER和CRY调制的结构机制。我们将首先基于功能和结构考虑生成各种功能相关的时钟蛋白构建体（目的1）。我们手头有过表达和纯化的重组构建体的结构化区域的个别时钟蛋白，并确定了复杂的结构的时钟：BMAL 1在我们的初步研究。我们将包括在我们的调查额外的功能域/时钟蛋白基序。CRY是CLOCK的主要阻遏物：BMAL 1。我们已经证明CRY在体外与CLOCK：BMAL 1形成稳定的复合物。在目的2中，我们将确定CRY：CLOCK：BMAL 1复合物的结构，以了解CRY如何与CLOCK：BMAL 1相互作用并抑制其反式激活功能。在最后一个目标（目标3），我们将研究PER与其他时钟组件，CRY：PER和PER：时钟：BMAL 1的相互作用，通过在体外和体内蛋白质-蛋白质相互作用测定，功能相关的复合物的结构测定，以及诱变和功能分析。我们希望了解CRY和PER在哺乳动物生物钟机制中的不同作用，以及它们如何与CLOCK：BMAL 1一起产生和维持哺乳动物的昼夜节律。

DESCRIPTION (provided by applicant): Circadian rhythm is a fundamental property of all eukaryotic and some prokaryotic organisms, allowing the organism to adapt its cellular metabolism and physiology to the ~24 hr light-dark cycle of the earth. In mammals, circadian rhythm is generated by an autoregulatory transcription-translation feedback loop, the core components of which are CLOCK, BMAL1, Cryptochrome (CRY), and Period (PER) proteins. The heterodimeric CLOCK:BMAL1 transcriptional factor activates the transcription of genes Cry and Per, and is in turn inhibited by their protein products CRY and PER, ultimately creating rhythmic gene expression patterns. Circadian clock is estimated to affect the expression of at least 10% of all genes in human genome, thus influences many aspects of metabolism, physiology, and behavior. Currently, little is known about molecular interactions between CLOCK:BMAL1 and its negative regulator CRY and PER at biochemical and structural levels, and how such interactions modulate the activity of CLOCK:BMAL1. The goal of the proposed study is to elucidate molecular interactions between CLOCK:BMAL1, CRY, and PER, through rigorous in vitro biochemical, biophysical and structural investigations, and to delineate the structural mechanisms by which the transcriptional function of CLOCK:BMAL1 is modulated by PER and CRY. We will first generate various functionally relevant constructs of clock proteins based on functional and structural considerations (Aim 1). We have in hand the over-expressed and purified recombinant constructs of the structured regions of individual clock proteins and have determined the complex structure of CLOCK:BMAL1 in our preliminary studies. We will include in our investigation additional functional domains/motifs of clock proteins. CRY is the main repressor of CLOCK:BMAL1. We have demonstrated that CRY forms stable complex with CLOCK:BMAL1 in vitro. In Aim 2, we will determine the structure of CRY:CLOCK:BMAL1 complex to learn how CRY interacts with and represses the transactivation function of CLOCK:BMAL1. In the last Aim (Aim 3), we will investigate the interactions of PER with other clock components, CRY:PER and PER:CLOCK:BMAL1, by in vitro and in vivo protein-protein interaction assays, structure determination of functionally relevant complexes, as well as mutagenesis and functional analysis. We hope to learn the distinct role of CRY and PER in the mammalian clock mechanism and how they work together with CLOCK:BMAL1 to generate and maintain circadian rhythm in mammals.