Post-translational control mechanisms of the circadian clock

描述（由申请人提供）：本提案旨在研究拟南芥中生物钟控制的三种不同的翻译后机制。首先是蛋白质磷酸化的作用，以及对关键时钟蛋白的活性、定位和稳定性的影响。拟南芥时钟中五个关键蛋白的稳健期特异性磷酸化引发了其在其活性，定位和周转中的重要性的问题。我们将重点关注拟南芥时钟中首次验证的磷酸化依赖性蛋白-蛋白相互作用，以开始了解TOC1和PRR3翻译后的动态。此外，我们提出了一种新的方法来发现新的激酶和磷酸酶作用于时钟蛋白。我们还建议开始对昼夜节律系统的这一部分进行建模，以解决不基于转录的时钟电路。二是伴侣蛋白在F-box蛋白ZEITLUPE成熟到其功能状态中所起的作用，这是维持稳健的昼夜节律振幅和周期所必需的。我们已经确定了两个新的组成部分，任何昼夜节律系统可能有助于ZTL成熟。通过对ZTL的影响，这两个成分间接调节昼夜节律系统。这两种蛋白在获得完全活性ZTL中的作用及其潜在的相互作用是潜在的问题。第三种机制是核孔在调节细胞核输入/输出mRNA和/或时钟基因蛋白中的作用。核孔是细胞核的看门人，所有转录因子和其他核功能调节因子都必须通过这个高度复杂的结构。所有真核生物的昼夜节律系统都涉及mRNA和蛋白质的核胞质穿梭，这可能在很大程度上有助于建立24小时分子周期性所需的时间延迟。我们已经确定了一种核孔成分，当缺乏时，它会减慢生物钟。了解这种延迟的分子基础将有助于更好地理解昼夜节律如何与细胞内运输联系在一起。以一种相关的方式，TOC1/PRR5相互作用似乎促进TOC1进入核，也可能用于招募激酶参与相互作用。这种相互作用的分子基础及其对周期的影响将使人们对生物钟中受调节的核进入的作用有更多的了解。虽然早期的时钟启发式模型侧重于转录/翻译反馈循环，但最近在所有昼夜节律系统中的发现都强调了这种观点的不足，以及翻译后机制如何对维持昼夜节律振荡做出重大贡献。下面描述的研究将有助于更好地理解昼夜节律钟，特别是振荡反馈系统。

DESCRIPTION (provided by applicant): This proposal is designed to investigate three different post-translational mechanisms of circadian clock control in Arabidopsis. The first is the role of protein phosphorylation, and the effect on activity, localization and stability of key clock proteins. Robust-phase specific phosphorylation of five key proteins in the Arabidopsis clock raises the question of its significance in their activity, localization and turnover. We will focus on the first validated phosphorylation-dependent protein-protein interaction in the Arabidopsis clock to begin to understand the dynamics of TOC1 and PRR3 post-translationally. Additionally, we propose a novel approach to discovering new kinases and phosphatases that act on clock proteins. We also propose to begin modeling this portion of the circadian system to address circuitry of the clock that is not based on transcription. The second is the effect chaperonins play in the maturation of the F-box protein ZEITLUPE to its functional state, necessary to the maintenance of robust circadian amplitude and period. We have identified two components new to any circadian system which likely contribute to ZTL maturation. Through effects on ZTL, these two components indirectly regulate the circadian system. The role of these two proteins in obtaining fully active ZTL, and their potential interaction is the underlying question addressed. The third mechanism is the role of the nuclear pore in the regulation of nuclear import/export of mRNA and/or protein of clock genes. The nuclear pore acts as a gatekeeper to the nucleus and all transcription factors and other regulators of nuclear function must pass through this highly complex structure. All eukaryotic circadian systems involve nucleocytoplasmic shuttling of mRNA and protein which may contribute substantially to establishing the timing delays necessary to establishing a 24 h molecular periodicity. We have identified a nuclear pore component that slows the circadian clock when absent. Understanding the molecular basis of this delay should lead to a greater understanding of how circadian timing is tied to intracellular transport. In a related way, the TOC1/PRR5 interaction appears to facilitate TOC1 nuclear entry and may also serve to recruit a kinase to the interaction. The molecular basis of this interaction and the effect on period will lead to increased understanding of the role of regulated nuclear entry in the clock. While the early heuristic models of the clock focused on transcription/translation feedback loops, recent findings across all circadian systems have highlighted the inadequacy of this view and how post- translational mechanisms contribute substantially to sustaining circadian oscillation. The studies described below will contribute to a greater understanding of circadian clock in particular, and to oscillatory feedback systems in general. PUBLIC HEALTH RELEVANCE: Circadian rhythms control a wide variety of processes in all eukaryotes, including recent linkages to the cell cycle and cancer. The fundamental organization of the clock as consisting of two or more, linked autoregulatory feedback loops is shared among all model systems, but the individual molecular players often vary among the kingdoms. Here post-translational mechanism of circadian clock control in Arabidopsis is investigated, with potential relevance to other eukaryotic oscillatory systems.