Molecular mechanisms underlying human circadian sleep disorders

项目摘要/摘要 生物钟出现故障的不良后果包括睡眠质量下降、睡眠质量差 表现，增加了短期内发生事故的风险，以及在 长期的。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 对于他们来说-是受到我们分子模型的不完备性和我们缺乏动物模型的限制。为 例如，许多有昼夜节律睡眠障碍的患者有每天变化的唤醒-睡眠周期 不可预知的方式。然而，没有动物模型可以模仿这种不稳定的节奏，直到我们的 实验室在过去的两年里开发出了一种。可能还有其他的人类昼夜节律紊乱 由于我们有限的一组动物模型远远不能与 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(SNPs)以阐明它们对生物钟功能的影响。因此，我们会加强 了解人类不同的时型和睡眠障碍并为发展铺平道路 有效的治疗方法。 具体目标1：确定可能与人类昼夜节律相关的PER基因变异 睡眠障碍。因为使用动物模型来概括所有已知的知识将是非常昂贵的 关于人类时钟基因的SNPs和突变，我们将首先描述大量重要的变异 细胞培养，使用U2OS和MEF，细胞系被广泛接受为昼夜节律的模型。我们的重点将是 因为PER1和PER2是最重要的哺乳动物基因，所以PER1和PER2是最重要的哺乳动物基因 在确定时钟的周期和相位时，每个基因的数百个变种已知存在。我们会 使用CRISPR/Cas9技术在时钟单元中复制不同的变体，我们将评估它们的 昼夜节律表型。我们已经确定了一个PER1缺失突变体，它揭示了一个新的对 磷酸化，这让我们提出了一个新的假说，即PER的磷酸化是如何动态调节的。我们 将检验这一假说，并继续研究翻译后调控的其他机制，这些机制对 钟。我们还将在小鼠身上复制一组精选的变异，用于活体测试。 具体目标2：确定PER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，生物钟依赖于核心时钟的节律 蛋白质Per，其蛋白酶体的降解受泛素化和去泛素化的调节。我们 建议对调节泛素化和降解的E3连接酶进行系统研究，以及 在细胞模型中测试它们的功能，以及候选脱泛素酶的功能。在目标1中，我们将 确定PER内的关键调控位点，而在目标2中，我们将确定参与该调控的关键酶。

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.