The Pathway that Sets the Cyanobacterial Circadian Clock

描述（由申请人提供）：从蓝藻到人类的不同生物体的细胞执行由内源性定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物化学事件，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，功能障碍会导致疾病。具体的目标解决的机制，这些方面的生物定时在蓝藻Synechococcus elongatus，最简单的模式生物已知有一个24小时的生物（昼夜节律）时钟。昼夜节律振荡器的组分是已知的（蛋白质KaiA、KaiB和KaiC），它们的结构已经被解决，并且KaiC的磷酸化的昼夜节律可以在体外重建。该生物体适合于遗传操作，并且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途径，以及使振荡器能够控制细胞功能的输出途径。该项目将提供深入了解时钟的每个方面的操作，它们的反馈和功能重叠，以及它们与细胞其他活动的整合。具体目标是：（1）定义输入途径组分的物理、生物化学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）定义刺激主要输出途径的KaiC的生物化学状态。质谱将用于确定由振荡器结合的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组分之间的物理相互作用。遗传和生物化学方法将确定负责新发现的KaiC刺激活性的因子和输入因子CikA的信号伴侣。在体外重构的振荡器将用于测试对输入和输出组分的循环KaiC磷酸化以及氧化还原调节的直接影响。生化测定和遗传报告系统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深入了解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功效。 公共卫生相关性：从蓝藻系统中吸取的教训将告知真核生物钟的操纵，包括病原体，营养源生物和人类的生物钟，以改善疾病预防和治疗，并提高健康。此外，该项目还将揭示可能在其他不同细菌（包括病原体）中发挥作用的新型信号转导机制。

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.