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）：昼夜节振荡器控制各种生物的每日节奏。在人类中，振荡器异常与睡眠障碍，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加速的生化过程不同，昼夜节律的通用特性是，它在所有生理温度下都保持恒定的速率（周期）。该过程称为温度补偿（TC），在维持自己体温的生物中是保守的。但是，尚不清楚TC的机制以及TC对昼夜节律健康的影响。在Neurospora中，最知名的时钟温度反应的生物涉及保守的PAS激酶（PSK）的活性。 PSK在高温下磷酸化阳性时钟成分WCC。缺乏PSK的细胞的时钟在高温下运行速度更快。酪蛋白激酶2（CK2）在高温下磷酸化并降低了阴性时钟成分FRQ的活性，而缺乏CK2的细胞在高温下运行较慢。但是，PSK和CK2缺陷的菌株是完全补偿的，这与其他因素改变时钟成分的观察结果一致。我们建议对TC进行测试和完善两个相互竞争的假设。 1）固有的温度依赖性增加正时钟和负时钟组件的水平相互平衡以保持一致的周期。 2）TC是一个新兴的网络，在该网络中，修改酶改变了时钟成分的活性，以补偿其在高温下观察到的水平升高。在AIM 1中，我们将测试TC所需的WCC的PSK磷酸化而不是其他目标。 WCC蛋白上的PSK靶序列将使用野生型（WT）中的质谱法（WT）和PSK损坏菌株在一天中的不同时间下确定。 PSK磷酸化位点将突变，并检查了损失TC的菌株。在同一实验中，将鉴定出WT细胞中时钟成分的其他潜在温度依赖性修饰，并具有对修饰位点突变的菌株，这些位点的突变因TC的损失而进行了。在AIM 2中，我们将通过识别依赖温度的周期修饰符来测试TC的模型。 TC因子缺失突变的组合将用于检验新兴网络假设。选择性操纵应变内WCC的水平（同时保持节奏性）以更改时钟组件的相对平衡将测试内在的TC假设。在AIM 3中，我们将确定调节时钟蛋白和PSK温度依赖性升高的机制。使用整个基因组核糖体分析与转录组分析相结合；我们将测试以下假设：生理温度升高特别影响时钟成分的翻译效率。这些研究共同将对我们对TC的理解产生重大影响，这是昼夜节律时钟的关键保守方面。