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: 10256761
• 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/10256761
• 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.

项目摘要/摘要 昼夜节律有故障的不利后果包括睡眠质量受损，很差 表现，并增加短期内发生事故的风险，以及代谢性疾病和癌症 长期。但是，我们对昼夜节律睡眠障碍的理解，因此我们有能力发展治疗 对于他们来说，受分子模型的不完整和动物模型死亡的限制。为了 例如，许多患有昼夜节律睡眠障碍的患者的睡眠周期每天在 不可预测的方式。然而，没有动物模型模仿如此不稳定的节奏直到我们 实验室在过去两年中开发了一个。可能还有其他人类昼夜节律疾病 由于我们有限的动物模型远远不与之匹配，因此无法识别或不理解 人类基因库的多样性。我们建议研究一组高度多样的突变和单核苷酸 多态性（SNP）阐明了它们对昼夜节律功能的影响。因此，我们会增强 了解人类的潜水员计时型和睡眠障碍，并为发展铺平道路 有效的治疗方法。 特定目的1：确定可能与人类昼夜节律有关的遗传变异 睡眠障碍。因为禁止使用动物模型概括所有已知的 SNP和人类时钟基因中的突变，我们将首先描述大量重要变体 细胞培养，使用U2OS和MEF，细胞系广泛接受为昼夜节律的模型。我们的重点意志 由于PER1和2是最重要的哺乳动物基因，因此在周期（每个）基因中成为SNP或突变 在确定时钟的周期和阶段，并且已知每个基因的数百个变体存在。我们将 使用CRISPR/CAS9技术在时钟单元中重现潜水员的变体，我们将评估它们 昼夜节律表型。我们已经确定了一个PER1缺失突变体，揭示了一个新的主题至关重要 磷酸化，导致我们对每磷酸化的动态调节方式进行了新的假设。我们 将检验这一假设，并继续研究翻译后调节的其他机制 钟。我们还将在小鼠中重现一组选择体内测试的变体。 特定目的2：确定稳健和定时蛋白酶体降解的关键调节剂。我们的 以前和正在进行的研究表明，昼夜节律取决于核心时钟的节奏 蛋白质及其蛋白酶体降解受泛素化和去泛素化的调节。我们 提议系统地搜索E3连接酶，该连接酶调节每个泛素化和降解，以及 测试其在细胞模型中的功能以及候选去泛酶的功能。在AIM 1中，我们将 确定PER内的关键监管站点，而在AIM 2中，我们将确定与该法规有关的关键酶。