Molecular mechanisms of mammalian circadian clock function - Renewal - 1

摘要 整个身体的生物钟驱动着数千个基因的有节奏的表达，导致 生物化学、生理和行为的节律。通过基因或基因改变昼夜节律 时差或倒班等环境干扰可能会产生深远的负面后果 并与肥胖、糖尿病、癌症、心血管疾病和精神疾病有关。在……里面 具体地说，生物钟控制着几乎每一条主要的代谢途径，从而实现最佳的 养分的典型循环有效性的利用。我们的工作主要集中在 了解哺乳动物生物钟的详细分子机制 机械和这些时钟控制有节奏的新陈代谢的机制。 根据目前的模型，这种时钟机制的核心部分是一个负反馈回路 由此转录因子异二聚体CLOCK/BMAL1驱动“CLOCK”的转录 与之相互作用的蛋白质周期(PER)1、PER 2、隐色素(CRY)1和CRY2 其他则抑制Clock/BMAL1的活性，从而抑制自身的合成。这是一样的 转录机制也驱动许多所谓的“时钟控制基因”的节律性表达。 这最终导致了全身组织的许多输出节律。其中一个时钟- 受控基因是Nocturin，我实验室的一个重点。我们已经在老鼠身上证明了这种基因的丢失 引起对饮食诱导的肥胖的抵抗，改变胆固醇和甘油三酯代谢的节律 并增加对炎症性挑战(如内毒素)的抵抗力。尽管Nocturin蛋白是 作为核糖核酸酶(CCR4死烯基酶)家族的一员，我们最近已经证明 Nocturin的底物不是RNA，而是NADP(H)。夜转素是一种NADP(H)磷酸酶， 将NADP(H)转换为NAD(H)。在这项提案中，我们寻求使用我们现有的新鼠标模型 了解夜翻素的组织特异性作用，并将两者的作用分开 异构体(一个线粒体，另一个主要结合在细胞膜上)。我们还将携带 体外代谢和代谢通量实验，以确定昼夜节律调节的影响 NADP(H)和NAD(H)水平。此外，我们还将验证夜曲灵小分子调节剂的有效性 我们已经在高通量筛选中发现了这些分子，并将使用这些分子来扰乱Nocturin 细胞和小鼠的活动。我们关于核心时钟中央机制的工作也将在#年继续进行。 拟议的资金期。我们已经解决了Clock/BMAL1和 Cry2/PER2复合体和这些数据使进化保守的 整个蛋白质的功能结构域，并通过以下方式揭示了对机制的更多见解 这些蛋白质在其中起作用并设定昼夜节律。在未来五年，我们将扩大 根据这些信息来确定这个时钟如何计时的原子细节。

Abstract Circadian clocks throughout the body drive rhythmic expression of thousands of genes, resulting in rhythms in biochemistry, physiology and behavior. Disruption of circadian clocks through genetics or environmental perturbations such as jet lag or shift-work, can have profound negative consequences and has been linked to obesity, diabetes, cancer, cardiovascular disease and mental illness. In particular, circadian clocks exert control over nearly every major metabolic pathway, allowing optimal utilization of typically cyclic availability of nutrients. Our work is focused generally on understanding the detailed molecular mechanisms of the mammalian circadian clock machinery and the mechanisms by which these clocks control rhythmic metabolism. According to the current model, the core part of this clock mechanism is a negative feedback loop whereby the transcription factor heterodimer CLOCK/BMAL1 drives transcription of the “clock” proteins PERIOD (PER) 1, PER 2, CRYPTOCHROME (CRY) 1 and CRY 2 which interact with each other to repress the activity of CLOCK/BMAL1, and thus their own synthesis. This same transcriptional mechanism also drives rhythmic expression of many so called “clock-controlled genes” that ultimately result in the many output rhythms in tissues throughout the body. One of these clock- controlled genes is Nocturnin, a focus of my laboratory. We have shown that loss of this gene in mice causes resistance to diet-induced obesity, alters rhythms in cholesterol and triglyceride metabolism and increases resistance to inflammatory challenges such as LPS. Although the Nocturnin protein is a member of a family of RNases (the CCR4 deadenylases), we have recently demonstrated that Nocturnin’s substrate is not RNA, but rather NADP(H). Nocturnin is an NADP(H) phosphatase, converting NADP(H) to NAD(H). In this proposal we seek to use new mouse models that we have developed to understand the tissue-specific role of Nocturnin and to disentangle the roles of the two isoforms (one mitochondrial, the other primarily bound to intracellular membranes). We will also carry out metabolomic and metabolic flux experiments to determine the effects of circadian regulation of NADP(H) and NAD(H) levels. In addition, we will validate small molecule modulators of Nocturnin that we have identified in a high throughput screen and will use these molecules to perturb Nocturnin activity in cells and mice. Our work on the central mechanism of the core clock will also continue in the proposed funding period. We have solved crystal structures for the CLOCK/BMAL1 and CRY2/PER2 complexes and these data have allowed the identification of evolutionarily conserved functional domains throughout the proteins and revealed additional insights into the mechanisms by which these proteins operate and set the circadian period. Over the next five years, we will expand on this information to determine the atomic details of how this clock keeps time.