Determining the Mechanism of Temperature Compensation of the Circadian Clock

确定昼夜节律时钟的温度补偿机制

• 批准号: 9061721
• 负责人: Deborah Bell-Pedersen
• 金额: $ 27.35万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9061721
• 关键词: Affect; Biochemical Process; Biological Assay; Body Temperature; Buffers; Cardiovascular Diseases; Cells; Circadian Rhythms; Clock protein; Collection; Complex; Coupled; Data; Defect; Deletion Mutation; Enzymes; Equilibrium; Feedback; Financial compensation; Gene Expression; Genes; Goals; Harvest; Health; High temperature of physical object; Human; Introns; Investigation; Knock-out; Malignant Neoplasms; Mass Spectrum Analysis; Messenger RNA; Metabolic syndrome; Modeling; Modification; Molecular; Mutate; Mutation; Neurospora; Neurospora crassa; Organism; Periodicity; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Process; Property; Proteins; RNA Splicing; Resources; Role; Running; Site; Sleep Disorders; Structure; Temperature; Testing; Time; Translations; Work; casein kinase II; cell type; circadian pacemaker; cold temperature; fitness; human disease; insight; mutant; phosphatidylinositol 3' -kinase-associated serine kinase; processing speed; protein complex; research study; response; ribosome profiling; transcription factor; transcriptome; whole genome

DESCRIPTION (provided by applicant): Circadian oscillators control daily rhythms in diverse organisms. In humans, abnormalities in the oscillator are associated with sleep disorders, cardiovascular disease, metabolic syndrome, and cancer. Unlike most biochemical processes that speed up as temperature rises, a universal property of the circadian clock is that it maintains nearly constant rate (period) at all physiological temperatures. This process, called temperature compensation (TC), is conserved in organisms that maintain their own body temperature. However, the mechanism of TC, and the impact of TC in circadian health is not known. In Neurospora, the organism in which temperature responses of the clock are best known, TC involves the activity of a conserved PAS kinase (PSK). PSK phosphorylates the positive-acting clock component WCC at high temperature. Cells that lack PSK have a clock that runs faster at high temperatures. Casein kinase 2 (CK2) phosphorylates and reduces the activity of the negative-acting clock component FRQ at high temperature, and cells lacking CK2 run slower at high temperatures. However, strains with defects in PSK and CK2 are fully compensated, consistent with observations that other factors modify the clock components. We propose to test, and refine, two competing hypotheses for TC. 1) An intrinsic temperature-dependent increase in the levels of the positive and negative clock components counter-balance each other to maintain consistent period. 2) TC is an emergent network in which period modifying enzymes alter the activities of the clock components to compensate for their observed increased levels at high temperature. In Aim 1, we will test the model that PSK phosphorylation of the WCC, as opposed to some other target, is necessary for TC. PSK target sequences on WCC proteins will be determined using mass spectroscopy in wild type (WT) and PSK-deletion strains at low and high temperature at different times of the day. PSK phosphorylation sites will be mutated, and the strains examined for loss of TC. In the same experiment, other potential temperature-dependent modifications of the clock components in WT cells will be identified, and strains with mutations of the modifications sites examined for loss of TC. In Aim 2, we will test the models for TC by identifying temperature-dependent period modifiers. Combinations of deletion mutations of the TC factors will be used to test the emerging network hypothesis. Selectively manipulating the levels of the WCC within strains (while maintaining rhythmicity) to change the relative balance of the clock components will test the intrinsic TC hypothesis. In Aim 3, we will determine the mechanism regulating a temperature-dependent increase in clock proteins and PSK. Using whole genome ribosome profiling coupled with transcriptome analyses; we will test the hypothesis that a physiological temperature increase specifically affects translation efficiency of the clock components. Together these studies will have a major impact on our understanding of TC, a key conserved aspect of circadian clocks.

描述（由申请人提供）：昼夜节律振荡器控制各种生物的日常节律。在人类中，振荡器的异常与睡眠障碍、心血管疾病、代谢综合征和癌症有关。与大多数随着温度升高而加速的生化过程不同，生物钟的一个普遍特性是，它在所有生理温度下都保持几乎恒定的速率（周期）。这个过程被称为温度补偿（TC），在维持自身体温的生物体中是保守的。然而，TC的机制以及TC对昼夜健康的影响尚不清楚。在神经孢子虫中，生物钟的温度反应最为人所知，TC涉及一种保守的PAS激酶（PSK）的活性。PSK在高温下磷酸化正作用时钟组件WCC。缺乏PSK的细胞有一个在高温下运行得更快的时钟。酪蛋白激酶2 （CK2）在高温下磷酸化并降低负作用时钟组分FRQ的活性，缺乏CK2的细胞在高温下运行较慢。然而，PSK和CK2缺陷的应变被完全补偿，这与其他因素改变时钟成分的观察结果一致。我们建议测试和完善两个相互竞争的TC假设。1)固有的温度依赖于正、负时钟组件水平的增加相互抵消，以保持一致的周期。2) TC是一个新兴的网络，其中周期修饰酶改变时钟组件的活动，以补偿它们在高温下观察到的增加的水平。在Aim 1中，我们将测试WCC的PSK磷酸化（而不是其他靶标）对TC是必要的模型。野生型（WT）和PSK缺失菌株在低温和高温下，在一天中的不同时间，使用质谱法测定WCC蛋白上的PSK靶序列。PSK磷酸化位点将发生突变，并检查菌株是否丢失TC。在相同的实验中，将鉴定WT细胞中时钟成分的其他潜在温度依赖性修饰，并检查修饰位点突变的菌株是否丢失TC。在目标2中，我们将通过识别与温度相关的周期修饰符来测试TC模型。TC因子的缺失突变组合将用于检验新兴的网络假设。有选择地操纵菌株内WCC的水平（同时保持节律性）来改变时钟组件的相对平衡将测试内在TC假设。在Aim 3中，我们将确定调节时钟蛋白和PSK的温度依赖性增加的机制。利用全基因组核糖体分析结合转录组分析；我们将检验生理温度升高具体影响时钟组件翻译效率的假设。总之，这些研究将对我们对昼夜节律的一个关键保守方面的理解产生重大影响。