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，使生物能够使其细胞代谢和生理适应地球约24小时的光暗循环。在哺乳动物中，昼夜节律是由一个自我调节的转录-翻译反馈回路产生的，其核心成分是CLOCK、BMAL1、Cryptochrome （CRY）和Period （PER）蛋白。异二聚体CLOCK:BMAL1转录因子激活Cry和Per基因的转录，并反过来被它们的蛋白产物Cry和Per抑制，最终形成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，从而影响新陈代谢、生理和行为的许多方面。目前，关于CLOCK:BMAL1与其负调节因子CRY和PER在生化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:BMAL1的活性，我们知之甚少。本研究的目的是通过严格的体外生化、生物物理和结构研究，阐明CLOCK:BMAL1、CRY和PER之间的分子相互作用，并描绘PER和CRY调节CLOCK:BMAL1转录功能的结构机制。我们将首先基于功能和结构考虑生成时钟蛋白的各种功能相关结构（目的1）。我们已经掌握了单个时钟蛋白结构区域的过表达和纯化重组结构，并在初步研究中确定了clock:BMAL1的复杂结构。我们将在我们的研究中包括时钟蛋白的其他功能域/基序。CRY是CLOCK:BMAL1的主要抑制因子。我们已经证明，CRY与CLOCK:BMAL1在体外形成稳定的复合物。在Aim 2中，我们将确定CRY:CLOCK:BMAL1复合物的结构，以了解CRY如何与CLOCK:BMAL1相互作用并抑制其交易功能。在最后一个目标（目标3）中，我们将通过体外和体内蛋白质-蛋白质相互作用测定、功能相关复合物的结构测定以及诱变和功能分析来研究PER与其他时钟成分CRY:PER和PER: clock:BMAL1的相互作用。我们希望了解CRY和PER在哺乳动物生物钟机制中的独特作用，以及它们如何与clock:BMAL1协同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.