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: 8840613
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8840613
• 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; Gene Expression Profile; Genes; Genome; 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; Relative (related person); Resources; Ribosomes; 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; targeted sequencing; transcription factor

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不同生物体的日常节律。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 的作用机制以及 TC 对昼夜节律健康的影响尚不清楚。在脉孢菌这种生物钟温度响应最为人所知的生物体中，TC 涉及保守的 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 的电池的时钟在高温下运行得更快。酪蛋白激酶 2 (CK2) 在高温下会磷酸化并降低负作用时钟成分 FRQ 的活性，缺乏 CK2 的细胞在高温下运行速度会变慢。然而，PSK 和 CK2 中存在缺陷的应变得到了完全补偿，这与其他因素修改时钟组件的观察结果一致。我们建议测试和完善 TC 的两个相互竞争的假设。 1) 正负时钟组件电平的固有温度依赖性增加相互抵消，以保持一致的周期。 2) TC 是一个新兴网络，其中周期修饰酶改变时钟组件的活动，以补偿其在高温下观察到的增加的水平。在目标 1 中，我们将测试以下模型：WCC 的 PSK 磷酸化（而不是其他一些靶标）对于 TC 是必需的。 WCC 蛋白上的 PSK 靶序列将在一天中不同时间的低温和高温下使用野生型 (WT) 和 PSK 缺失菌株的质谱法进行测定。 PSK 磷酸化位点将发生突变，并检查菌株是否有 TC 丢失。在同一实验中，将鉴定 WT 细胞中时钟组件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有 TC 丢失。在目标 2 中，我们将通过识别与温度相关的周期调节因子来测试 TC 模型。 TC 因子的缺失突变组合将用于测试新兴的网络假设。有选择地操纵菌株内 WCC 的水平（同时保持节律性）以改变时钟成分的相对平衡将检验内在的 TC 假设。在目标 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。使用全基因组核糖体分析与转录组分析相结合；我们将测试生理温度升高具体影响时钟组件的转换效率的假设。这些研究将对我们对 TC（生物钟的一个关键保守方面）的理解产生重大影响。