Clock Mechanism Underlying Drosophila Rhythmic Behavior

描述（由申请人提供）：人类昼夜节律（~24小时）计时系统的故障和/或“时钟”基因的突变与许多障碍和疾病有关，包括情感障碍、慢性睡眠问题、一系列代谢综合征，甚至对癌症的易感性。基于许多证据，现在已经确定，一个或多个中心时钟蛋白磷酸化状态的时间特异性变化是设定生物钟节奏的关键状态变量。在动物中，PERIOD（PER）蛋白是生物钟的组成部分，充当主要的“磷酸定时器”。研究表明，人类PER 2上的磷酸化位点或磷酸化PER的激酶的突变是几种家族性晚期睡眠综合征（FASPS）的基础，这突出了PER磷酸化对人类健康的重要性。PER蛋白的一个关键功能是，它们与其他核心时钟蛋白一起参与生成一个基于转录负反馈环的多组分生化振荡器，该振荡器不仅使时钟基因表达的每日周期永久化，而且还驱动约10%的细胞转录本的节律表达，这最终成为许多观察到的昼夜节律的基础。PER蛋白在周期性基因表达中起着关键作用，它以阶段特异性的方式作为“支架”将多亚基“阻遏物”复合物募集到中央时钟转录因子。PER磷酸化的一天中的时间特异性变化对于产生昼夜节律基因表达是至关重要的，其通过调节其丰度、进入核的时间、在核中的持续时间和可能的阻遏物效力来限制PER在日常周期中何时参与转录阻遏。 许多核心时钟蛋白，如中央转录因子，被磷酸化，尽管这些磷酸化事件的功能尚不清楚。本文提出的工作将通过使用遗传上易处理的模式生物黑腹果蝇来增加对昼夜节律机制的生化基础的重要新见解：1）鉴定关键时钟蛋白上的磷酸化位点，并确定它们的生物化学和生理学作用，2）鉴定相关激酶并确定它们在时钟机构中的作用，以及3）分离天然时钟复合物，识别组成因素并确定它们在昼夜节律产生中的作用。一个更全面的看法，磷网络运行在钟表及其相互关系与时钟复杂的组件/功能将达到。

DESCRIPTION (provided by applicant): Malfunctions in the human circadian (~24 hr) timing system and/or mutations in 'clock' genes are implicated in many disorders and diseases including affective disorders, chronic sleep problems, a range of metabolic syndromes and even susceptibility to cancer. Based on many lines of evidence it is now established that time-of-day specific changes in the phosphorylation state of one or more central clock proteins is the key state variable setting the pace of circadian clocks. In animals, PERIOD (PER) proteins are the clock components behaving as the primary 'phospho-timer.' The importance of PER phosphorylation to human health is highlighted by studies showing that mutations in either a phosphorylation site on human PER2 or a kinase that phosphorylates PER underlie several familial advanced sleep phase syndromes (FASPS). A critical function of PER proteins is that they participate with other core clock proteins to generate a multi-component biochemical oscillator based on transcriptional negative feedback loops that not only perpetuate daily cycles in the expression of clock genes but also drive rhythmic expression of about 10 percent of a cell's transcripts, which ultimately underlies many of the observed circadian rhythms. PER proteins play a critical role in cyclical gene expression by acting in a phase-specific manner as 'scaffolds' to recruit multi-subunit 'repressor' complexes to central clock transcription factors. Time-of-day specific changes in PER phosphorylation are critical for generating circadian gene expression by limiting when in a daily cycle PER engages in transcriptional repression by regulating its abundance, timing of nuclear entry, duration in the nucleus and possibly repressor potency. Many of the core clock proteins, such as the central transcription factors, are phosphorylated, although the functions of these phosphorylation events are not clear. The work proposed herein will add significant new insights into the biochemical basis underlying circadian machineries by using the genetically tractable model organism Drosophila melanogaster to: 1) identify phosphorylation sites on key clock proteins, and determine their biochemical and physiological roles, 2) identify the relevant kinases and determine their roles in the clockworks, and 3) isolate native clock complexes, identify constituent factors and determine their role(s) in circadian rhythm generation. A more comprehensive view of the phospho-networks operating in the clockworks and its inter-relationships with clock complex assembly/function will be attained.