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：
8422664
负责人：
JOSEPH S TAKAHASHI
金额：
$ 29.09万
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/8422664
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某些原核生物的基本特性，使该生物可以使其细胞代谢和生理学适应地球的〜24小时灯光周期。在哺乳动物中，昼夜节律是由自动调节转录 - 翻译回路产生的，其核心成分是时钟，bmal1，cryptochrome（cry）和周期（per）蛋白质。异二聚体时钟：BMAL1转录因子激活基因哭泣和Per的转录，而其蛋白质产物哭泣和Per又抑制了基因的转录，最终会产生节奏基因表达模式。据估计，昼夜节律时钟会影响人类基因组中所有基因的至少10％的表达，从而影响代谢，生理和行为的许多方面。当前，对于时钟：BMAL1及其负调节剂的分子相互作用以及在生化和结构水平上的PER，以及这种相互作用如何调节时钟的活性：BMAL1之间的分子相互作用知之甚少。拟议的研究的目的是通过严格的体外生化，生物物理和结构研究来阐明时钟：bmal1，cry和per之间的分子相互作用，并描述了按每个和哭泣调节时钟：bmal1的转录功能的结构机制。我们将根据功能和结构考虑（AIM 1）首先生成各种功能相关的时钟蛋白构建体。在我们的初步研究中，我们手头上有过表达和纯化的重组结构的重组构建体，并确定了时钟的复杂结构：bmal1。我们将在调查中包括时钟蛋白的其他功能域/基序。哭泣是时钟的主要阻遏物：bmal1。我们已经证明了哭泣形成稳定的复合物与时钟：BMAL1体外。在AIM 2中，我们将确定哭泣的结构：时钟：BMAL1复合物，以了解哭泣的相互作用并抑制时钟：BMAL1的反式激活函数。在最后一个目标（AIM 3）中，我们将通过体外和体内蛋白质 - 蛋白质相互作用测定法，功能相关的复合物以及诱变和功能分析的结构确定Per与其他时钟组件的相互作用，cry：cry：per：per：per：percem：clock：bmal1。我们希望学习哭泣和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。