The Pathway that Sets the Cyanobacterial Circadian Clock

描述(申请人提供)：不同生物的细胞，从蓝藻到人类，执行由内源性计时机制驱动的生理过程的时间程序。该项目的总体目标是了解生化事件，使细胞能够跟踪时间，根据时间程序执行活动，并使内部时钟与外部太阳周期同步。这些过程对健康至关重要，而功能障碍会导致疾病。具体目标是解决蓝藻聚球藻生物计时的每一个方面的机制，聚球藻是已知的最简单的模式生物，具有24小时的生物(昼夜节律)时钟。昼夜节律振荡器的组成是已知的(蛋白KaIa、Kaib和KaiC)，它们的结构已经被解决，并且在体外可以重建KaiC磷酸化的昼夜节律。生物体可以接受遗传操作，其基因表达的昼夜节律可以通过荧光素酶报告基因可视化。所有生物体群体的时钟机制在操作上分为振荡器，使振荡器与太阳日同步并在不同分子振荡器之间提供内部协调的输入路径，以及使振荡器能够控制细胞功能的输出路径。该项目将深入了解时钟的每一个方面的运作、它们的反馈和功能重叠，以及它们与该小组其他活动的结合。具体目标是：(1)确定输入途径组分的物理、生化和氧化还原相互作用，(2)评估输入和输出组分对振荡器活性的调节，以及(3)确定刺激主要输出途径的KaiC的生化状态。质谱学将被用来确定与振荡器结合的苯醌氧化还原辅因子的身份和活性，并定义输入、输出和振荡器蛋白质以及其他细胞成分之间的物理相互作用。遗传和生化方法将确定负责新发现的KaiC刺激活性的因素和输入因子Cika的信号伙伴。在体外重组的振荡器将被用来测试输入和输出成分的循环KaiC磷酸化以及氧化还原调节的直接影响。生化分析和遗传报告系统都将用于确定触发下游信号系统的振荡器的生化状态。对昼夜节律振荡器及其与细胞代谢的整合的洞察将为医学进步提供信息，例如了解昼夜节律与代谢综合征的联系，以及提高药物干预的有效性。 公共卫生相关性：从蓝藻系统中吸取的经验教训将有助于操纵真核生物钟，包括病原体、营养源生物体和人类的生物钟，以改进疾病预防和治疗，并增进健康。此外，该项目将揭示新的信号转导机制，这些机制可能在其他不同的细菌中运行，包括病原体。

DESCRIPTION (provided by applicant): Cells of diverse organisms, from cyanobacteria to humans, execute temporal programs of physiological processes that are driven by endogenous timing mechanisms. The overall goal of this project is to understand the biochemical events that allow a cell to keep track of time, execute activities according to a temporal program, and synchronize the internal clock with the external solar cycle. These processes are crucial to wellness, and dysfunction results in disease. The specific aims address the mechanisms of each of these aspects of biological timing in the cyanobacterium Synechococcus elongatus, the simplest model organism known to have a 24-h biological (circadian) clock. The components of the circadian oscillator are known (proteins KaiA, KaiB, and KaiC), their structures have been solved, and the circadian rhythm in phosphorylation of KaiC can be reconstituted in vitro. The organism is amenable to genetic manipulation and its circadian rhythms of gene expression can be visualized through luciferase reporter genes. Clock mechanism across all groups of organisms is operationally divided into the oscillator, the input pathways that synchronize the oscillator with the solar day and provide internal coordination among distinct molecular oscillators, and the output pathways that enable the oscillator to control cellular functions. This project will provide insight into the operation of each of these aspects of the clock, their feedbacks and functional overlaps, and their integration with other activities of the cell. The specific aims will: (1) Define the physical, biochemical, and redox interactions of input pathway components, (2) Assess the modulation of oscillator activity by input and output components, and (3) Define the biochemical states of KaiC that stimulate the major output pathway. Mass spectrometry will be used to determine the identity and activity of quinone redox cofactors that are bound by the oscillator, and to define the physical interactions among input, output, and oscillator proteins, as well as other cellular components. Genetic and biochemical methods will identify the factor responsible for a newly discovered KaiC-stimulating activity and the signaling partner of the input factor CikA. Oscillators reconstituted in vitro will be used to test the direct influence on cyclic KaiC phosphorylation of input and output components, and of redox modulation. Both biochemical assays and a genetic reporting system will be used to determine the biochemical state of the oscillator that triggers the downstream signaling system. Insights into a circadian oscillator and its integration with cellular metabolism will inform medical advances, such as understanding the circadian link to metabolic syndrome and improving the efficacy of pharmaceutical interventions. PUBLIC HEALTH RELEVANCE: Lessons learned from the cyanobacterial system will inform the manipulation of eukaryotic clocks, including those of pathogens, nutritional source organisms, and of humans, to improve disease prevention and treatment, and to enhance wellness. Furthermore, the project will reveal novel signal transduction mechanisms that are likely to operate in other diverse bacteria, including pathogens.