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不同生物体的每日节律。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物化学过程不同，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的机制，以及TC对昼夜健康的影响尚不清楚。在脉孢菌中，生物钟的温度响应是最为人所知的，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成分FRQ的活性，缺乏CK2的细胞在高温下运行较慢。然而，在PSK和CK2中具有缺陷的应变被完全补偿，与其他因素修改时钟分量的观察一致。我们建议测试，并完善，两个相互竞争的假设TC。1)正时钟分量和负时钟分量的电平的固有温度依赖性增加彼此抵消以维持一致的周期。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水平。在目标1中，我们将测试WCC的PSK磷酸化，而不是其他一些目标，是TC所必需的模型。将在一天中的不同时间在低温和高温下使用质谱法在野生型（WT）和PSK缺失菌株中测定WCC蛋白上的PSK靶序列。PSK磷酸化位点将被突变，并检查菌株的TC损失。在同一实验中，将鉴定WT细胞中时钟组分的其他潜在温度依赖性修饰，并检查具有修饰位点突变的菌株的TC损失。在目标2中，我们将通过识别温度依赖的周期修正因子来测试TC模型。TC因子的缺失突变组合将用于检验新兴网络假设。选择性地操纵品系内的WCC水平（同时保持节律性）以改变生物钟组分的相对平衡将测试内在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加上转录组分析，我们将测试的假设，即生理温度的增加，特别是影响翻译效率的时钟组件。这些研究将对我们理解TC产生重大影响，TC是生物钟的一个关键保守方面。