Molecular and cellular mechanisms of circadian timekeeping in a prokaryote model

原核生物模型中昼夜节律的分子和细胞机制

基本信息

批准号：
9900016
负责人：
SUSAN S GOLDEN
金额：
$ 59.42万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-04 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9900016
关键词：
Address Affect Anacystisnidulans Animal Model Animals Bacteria Biochemical Biological Clocks Biological Models Biological Rhythm Cardiovascular Diseases Cell Cycle Cell division Cells Circadian Dysregulation Circadian Rhythms Clock protein Cues Cyanobacterium Cytokinesis Disease Environment Eukaryota Exhibits Functional disorder Gene Expression Genetic Health Human In Vitro Inherited Interferometry Kinetics Label Malignant Neoplasms Mammalian Cell Mammals Measurement Mental disorders Metabolic Metabolic syndrome Modeling Molecular Mutation Organism Output Pathway interactions Periodicity Phenotype Physiological Preparation Prokaryotic Cells Property Protein Biochemistry Proteins Proteomics Sampling Sleep Disorders System Time circadian circadian pacemaker fitness human pathogen in vivo insight protein biomarkers public health relevance reconstitution transcription factor

项目摘要

DESCRIPTION (provided by applicant): This project addresses two fundamental questions: how does a circadian clock function at the molecular level as a timekeeping mechanism, and how is it integrated at the cellular level to control activities such as gene expression and cell division? The circadian clock is an oscillatory timer that drives 24-h rhythms of biological activities in diverse organisms from bacteria to humans, and disruptions in its underlying molecular mechanism adversely affect fitness. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. Despite different strategies for timekeeping that have evolved between cyanobacteria and mammals, the circadian clock of the cyanobacterium Synechococcus elongatus generates bona fide circadian rhythms of genetic, physiological, and metabolic activities that fulfill all criteria that define circadian clocks in eukaryotes. A quantitative, systems- level, biochemical understanding is attainable for the circadian clock of S. elongatus, whose fundamental circadian oscillator can be reconstituted in vitro with three proteins, KaiA, KaiB, and KaiC. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. Moreover, as is true in mammalian cells, the circadian clock of S. elongatus controls the timing of cell division. This project will leverage recent conceptual and technical advances to determine the mechanism of the timekeeping system with unprecedented clarity, understand how the clock controls activities in the cell, and elucidate how a sense of time is inherited when cells divide. The discoveries that KaiB refolds as part of the timekeeping mechanism and becomes a connector between oscillator and output pathways, and that metabolites are sampled by oscillator proteins to set the clock with local time, will enable establishment of a more complete in vitro clock that exhibits rhythmic output relevant for control of gene expression. Using this preparation, and kinetics measurements of partner interactions from BioLayer Interferometry, the project will quantify the steps that contribute to timekeeping, synchronization, and rhythmic output. Analysis in vivo of mutations that alter such interactions will tie specific steps to clock functions. The biochemical basis of interactions between two transcription factors that integrate temporal and environmental cues will be clarified. Time-lapse measurements of dividing cells that carry fluorescently labeled clock proteins and markers of the circadian cycle, in genetic backgrounds that are proficient or deficient in clock-control of cell division, will provide insight into how clock components are inherited with the correct timestamps. Proteomic approaches will identify partners responsible for localization of clock components within the cell and the ability of the clock to allow or disalow cytokinesis. Together, these approaches will elucidate clock mechanisms and the relationship between the circadian and cell division cycles.

描述（由适用提供）：该项目解决了两个基本问题：昼夜节律在分子水平上如何作为计时机制发挥作用，以及如何在细胞水平集成以控制基因表达和细胞分裂等活动？昼夜节律时钟是一个振荡的计时器，它在从细菌到人类到人类的潜水生物体中驱动生物活性的24小时节奏，并且其潜在的分子机制的破坏对适应性产生了不利影响。人类的时钟功能障碍与多种健康状况有关，例如心血管疾病，癌症，代谢综合征，精神疾病和睡眠障碍。尽管在蓝细菌和哺乳动物之间发展了不同的计时策略，但cyanobacterium synechococcus elongatus的昼夜节律产生了真正的遗传，生理和代谢活动的昼夜节律节奏，这些活动符合所有标准，这些标准符合Eukaryotes中所有定义昼夜节律时钟的标准。对于S.昼夜节律，可以获得定量的，系统水平的生化理解。 Elongatus，其基本的昼夜节律振荡器可以在体外与三种蛋白（Kaia，Kaib和Kaic）重新结构。在这种遗传上的模型生物中，可以系统地改变时钟蛋白的物理和生化特性，并追踪这些变化从其代理效应（通过蛋白质交互网络）到表达的昼夜节律表型的影响。此外，正如哺乳动物细胞所示一样，弹性链球菌的昼夜节律控制了细胞分裂的时间。该项目将利用最新的概念和技术进步来以前所未有的清晰度确定计时系统的机制，了解时钟如何控制细胞中的活动，并阐明当细胞分裂时如何继承时间感。 KAIB反映为计时机制的一部分，并成为振荡器和输出途径之间的连接器的发现，并且由振荡器蛋白采样代谢物以在当地时间设置时钟，将使您能够建立一个更完整的体外时钟，该时钟表现出与控制基因表达相关的节奏输出。使用此准备工作，以及从生物层干涉法的伴侣相互作用的动力学测量，该项目将量化有助于计时，同步和节奏输出的步骤。在体内分析改变这种相互作用的突变将将特定步骤绑定到时钟函数。将阐明两个转录因子之间相互作用的生化基础，这些转录因子将阐明。在精通或不足的细胞分裂时钟对照的遗传背景下，携带荧光标记的时钟蛋白和标记的分裂细胞的延时测量将提供有关如何用正确的时间戳记遗传的时钟成分的洞察力。蛋白质组学方法将确定负责将时钟组件定位的合作伙伴以及时钟允许或失去细胞因子的能力。这些方法一起将阐明时钟机制以及昼夜节律周期之间的关系。