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生物钟控制的三种不同翻译后机制。首先是蛋白磷酸化的作用，以及对关键时钟蛋白的活性、定位和稳定性的影响。拟南芥生物钟中五个关键蛋白的健壮期特异性磷酸化引起了人们对其活性、定位和周转的重要性的质疑。我们将集中在第一个验证的磷酸化依赖的蛋白质-蛋白质相互作用在拟南芥时钟开始了解TOC 1和PRR 3的动力学post-discriminatively。此外，我们提出了一种新的方法来发现新的激酶和磷酸酶的时钟蛋白的作用。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。 第二个是伴侣蛋白在F-box蛋白ZEITLUPE成熟到其功能状态中发挥的作用，这是维持强大的昼夜节律幅度和周期所必需的。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，这两种成分间接调节昼夜节律系统。这两种蛋白质在获得完全活性的ZTL中的作用，以及它们之间潜在的相互作用是要解决的根本问题。 第三种机制是核孔在调节时钟基因mRNA和/或蛋白质的核输入/输出中的作用。核孔作为细胞核的看门人，所有转录因子和其他核功能调节因子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和蛋白质的核质穿梭，这可能对建立24小时分子周期所需的时间延迟有很大贡献。我们已经确定了一个核孔组成部分，减缓生物钟时缺席。了解这种延迟的分子基础应该会导致更好地理解昼夜节律的时间是如何与细胞内运输。在一个相关的方式，TOC 1/PRR 5的相互作用似乎有助于TOC 1核进入，也可能有助于招募激酶的相互作用。这种相互作用的分子基础和对周期的影响将导致对时钟中受调节的核进入的作用的更多理解。 虽然早期的启发式模型的时钟集中在转录/翻译反馈回路，最近的研究结果在所有昼夜节律系统强调了这种观点的不足，以及翻译后机制如何大大有助于维持昼夜节律振荡。下面描述的研究将有助于更好地理解生物钟，特别是振荡反馈系统。 公共卫生相关性：昼夜节律控制着所有真核生物中的各种过程，包括最近与细胞周期和癌症的联系。生物钟的基本结构是由两个或多个相互连接的自动调节反馈回路组成，这在所有模型系统中都是共享的，但在不同的王国中，个体分子的参与者往往是不同的。在这里，拟南芥的生物钟控制的翻译后机制进行了调查，与其他真核生物振荡系统的潜在相关性。

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.