Molecular interactions of mammalian circadian clock proteins

哺乳动物生物钟蛋白的分子相互作用

基本信息

批准号：
8692928
负责人：
JOSEPH S TAKAHASHI
金额：
$ 30.21万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8692928
关键词：
ARNT gene Address Affect Affinity BMAL1 protein Back Behavior Binding Biochemical Biological Assay Cardiovascular Diseases Cells Circadian Rhythms Clock protein Complex Crying Crystallization DNA Binding Diabetes Mellitus Disease Feedback Fluorescence Foundations Gene Expression Profile General Transcription Factors Genes Genetic Transcription Goals Hand Health Helix-Turn-Helix Motifs Hour Human Human Genome In Vitro Individual Insecta Investigation Jet Lag Syndrome Knowledge Lead Learning Length Light Malignant Neoplasms Mammals Mediating Medicine Mental Depression Metabolism Methods Molecular Molecular Conformation Mus Mutagenesis Obesity Organism Physiology Plasma Play Property Protein Family Proteins Recombinants Research Role Science Sleeplessness Structure Surface System Transactivation Transcription Coactivator Translations Work activating transcription factor addiction arm base circadian pacemaker cryptochrome effective therapy feeding in vivo protein complex protein protein interaction public health relevance research study three dimensional structure

项目摘要

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.

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，使生物体能够使其细胞代谢和生理学适应地球约 24 小时的光暗周期。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、BMAL1、隐花色素 (CRY) 和周期 (PER) 蛋白。异二聚体 CLOCK:BMAL1 转录因子激活 Cry 和 Per 基因的转录，反过来又被它们的蛋白质产物 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% 的基因表达，从而影响新陈代谢、生理和行为的许多方面。目前，人们对 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，并描述 CLOCK:BMAL1 转录功能受PER 和 CRY。我们将首先根据功能和结构考虑生成各种功能相关的时钟蛋白构建体（目标 1）。我们手头有单个时钟蛋白结构区域的过表达和纯化重组构建体，并在我们的初步研究中确定了 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 的主要阻遏蛋白。我们已经证明 CRY 在体外与 CLOCK:BMAL1 形成稳定的复合物。在目标 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 的反式激活功能相互作用并抑制 CLOCK:BMAL1 的反式激活功能。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确定，研究 PER 与其他时钟组件 CRY:PER 和 PER:CLOCK:BMAL1 的相互作用，以及诱变和功能分析。我们希望了解 CRY 和 PER 在哺乳动物时钟机制中的独特作用，以及它们如何与 CLOCK:BMAL1 一起工作以产生和维持哺乳动物的昼夜节律。