Molecular mechanisms underlying human circadian sleep disorders

人类昼夜节律睡眠障碍的分子机制

• 批准号: 10006843
• 负责人: CHOOGON LEE
• 金额: $ 30.32万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-04 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006843
• 关键词: Accidents; Advanced Sleep Phase Syndrome; Affect; Alleles; Amino Acids; Animal Model; Biological Assay; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell model; Cells; Circadian Rhythms; Clinical; Clock protein; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Deubiquitination; Dimerization; Disease; Enzymes; Equilibrium; Event; Gene Pool; Genes; Genetic; Genetic Variation; Human; Human Genome; Individual; Kinetics; Laboratories; Lead; Length; Libraries; Link; Malignant Neoplasms; Mammals; Mediating; Metabolic Diseases; Missense Mutation; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Names; Pathologic; Patients; Performance; Performance at work; Periodicity; Phase; Phenotype; Phosphorylation; Phosphotransferases; Physiology; Post-Translational Regulation; Process; Proteins; Reaction; Regulation; Reporter; Risk; Role; S Phase; Single Nucleotide Polymorphism; Site; Sleep Disorders; Sleep Wake Cycle; System; Testing; Ubiquitination; Variant; Work; Yeasts; adverse outcome; beta-Transducin Repeat-Containing Proteins; circadian; circadian pacemaker; critical period; effective therapy; falls; in vivo evaluation; molecular modeling; mouse model; mutant; novel; polymerization; repaired; screening; sleep quality; trafficking; ubiquitin-protein ligase

Project Summary/Abstract Adverse consequences from having a faulty circadian clock include compromised sleep quality, poor performance, and increased risk for accidents in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian sleep disorders—and thus our ability to develop treatments for them—is limited by the incompleteness of our molecular models and our dearth of animal models. For example, many patients with circadian sleep disorders have wake-sleep cycles that shift daily in an unpredictable manner. Yet there were no animal models that emulated such unstable rhythms until our laboratory developed one in the last two years. There may be other human circadian disorders that go unrecognized or are poorly understood because our limited set of animal models fall far short of matching the diversity of the human gene pool. We propose to study a highly diverse set of mutations and single-nucleotide polymorphisms (SNPs) to elucidate their effect on circadian clock function. We would thus enhance the understanding of diverse chronotypes and sleep disorders in humans and pave the way for developing effective treatments. Specific Aim 1: Identify genetic variations in PER that may be associated with human circadian sleep disorders. Because it would be prohibitively expensive to use animal models to recapitulate all known SNPs and mutations in human clock genes, we will first characterize a large number of important variants in cell culture, using U2OS and MEFs, cell lines widely accepted as models for circadian rhythms. Our focus will be on SNPs or mutations in Period (Per) genes because Per1 and 2 are the most important mammalian genes in determining the clock’s period and phase, and hundreds of variants of each gene are known to exist. We will use the CRISPR/Cas9 technology to reproduce diverse variants in clock cells, and we will evaluate their circadian phenotypes. We have already identified a Per1 deletion mutant revealing a novel motif critical for phosphorylation, leading us to a novel hypothesis for how PER phosphorylation is dynamically regulated. We will test this hypothesis and continue to study other mechanisms of posttranslational regulation critical for the clock. We will also reproduce a select set of variants in mice for in vivo testing. Specific Aim 2: Identify key regulators of robust and timed proteasomal degradation of PER. Our previous and ongoing studies suggest that the circadian clock is dependent on rhythms of the core clock protein PER, and its proteasomal degradation is regulated by both ubiquitination and deubiquitination. We propose to conduct a systematic search for E3 ligases that regulate PER ubiquitination and degradation, and to test their function in cell models, along with the functions of candidate deubiquitnases. In Aim 1, we will identify key regulatory sites within PER, while in Aim 2, we will identify key enzymes involved in that regulation.

项目概要/摘要 生物钟错误带来的不良后果包括睡眠质量下降、睡眠质量差、 性能，短期内发生事故的风险增加，以及代谢性疾病和癌症的风险增加 长期。然而，我们对昼夜节律睡眠障碍的理解以及我们开发治疗方法的能力 对他们来说——受到我们分子模型的不完整性和动物模型的缺乏的限制。为了 例如，许多患有昼夜节律睡眠障碍的患者的觉醒-睡眠周期每天都会发生变化。 不可预测的方式。然而，直到我们发现之前，还没有动物模型能够模拟这种不稳定的节律。 实验室在过去两年开发了一种。可能还有其他人类昼夜节律紊乱 未被识别或了解甚少，因为我们有限的动物模型远不能匹配 人类基因库的多样性。我们建议研究一组高度多样化的突变和单核苷酸 多态性（SNP）来阐明它们对生物钟功能的影响。因此，我们将加强 了解人类不同的时间型和睡眠障碍，并为发展铺平道路 有效的治疗。 具体目标 1：识别可能与人类昼夜节律相关的 PER 遗传变异 睡眠障碍。因为使用动物模型来重现所有已知的情况会非常昂贵 人类时钟基因的 SNP 和突变，我们将首先表征大量重要的变异 细胞培养，使用 U2OS 和 MEF，细胞系被广泛接受为昼夜节律模型。我们的重点将 位于周期 (Per) 基因的 SNP 或突变上，因为 Per1 和 2 是最重要的哺乳动物基因 确定时钟的周期和相位，并且每个基因已知存在数百种变体。我们将 使用CRISPR/Cas9技术在时钟细胞中复制多种变异，我们将评估它们 昼夜节律表型。我们已经鉴定出 Per1 缺失突变体，揭示了一个对 磷酸化，使我们对 PER 磷酸化如何动态调节提出了一个新的假设。我们 将测试这一假设并继续研究对翻译后调控至关重要的其他机制 钟。我们还将在小鼠中复制一组选定的变体以进行体内测试。 具体目标 2：确定 PER 强力且定时的蛋白酶体降解的关键调节因子。我们的 先前和正在进行的研究表明，生物钟取决于核心时钟的节律 蛋白质 PER 及其蛋白酶体降解受到泛素化和去泛素化的调节。我们 提议对调节 PER 泛素化和降解的 E3 连接酶进行系统搜索，以及 测试它们在细胞模型中的功能以及候选去泛素酶的功能。在目标 1 中，我们将 确定 PER 内的关键调节位点，而在目标 2 中，我们将确定参与该调节的关键酶。