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(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的作用机制以及TC对昼夜节律健康的影响尚不清楚。在脉孢子虫中，生物钟的温度反应是最已知的，TC涉及一种保守的PAS激酶(PSK)的活性。PSK在高温下使正作用的时钟成分WCC磷酸化。缺乏PSK的细胞有一个在高温下运行更快的时钟。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，PSK和CK2中有缺陷的菌株得到了完全补偿，这与观察到的其他因素改变时钟分量的情况一致。我们建议对TC的两个相互竞争的假设进行测试和提炼。1)正负时钟分量水平的内在温度依赖增加相互抵消，以维持一致的周期。2)TC是一个紧急网络，在这个网络中，周期修饰酶改变时钟成分的活性，以补偿它们在高温下观察到的水平上升。在目标1中，我们将测试这一模型，即相对于其他目标，WCC的PSK磷酸化是TC所必需的。WCC蛋白上的PSK靶序列将在一天中的不同时间，在低温和高温下，利用质谱学在野生型(WT)和PSK缺失菌株中进行测定。PSK磷酸化位点将发生突变，并检查菌株的TC丢失情况。在同一实验中，将鉴定WT细胞中其他潜在的与温度有关的时钟成分的修饰，并检查具有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的模型。TC因子的缺失突变组合将被用来检验新兴网络假说。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假说。在目标3中，我们将确定调节依赖于温度的时钟蛋白和PSK增加的机制。使用全基因组核糖体图谱与转录组分析相结合的方法，我们将检验这一假设，即生理温度上升特别影响时钟组件的翻译效率。总而言之，这些研究将对我们对TC的理解产生重大影响，TC是生物钟的一个关键保守方面。