Molecular mechanisms of mammalian circadian clock function - Renewal - 1

摘要 整个身体的生物钟驱动着成千上万个基因的有节奏的表达， 生物化学、生理学和行为的节奏。通过遗传学或生物钟的破坏 环境干扰，如时差或轮班工作，可能会产生深远的负面影响 并与肥胖、糖尿病、癌症、心血管疾病和精神疾病有关。在 特别是，生物钟几乎控制着每一个主要的代谢途径， 利用养分的典型循环可用性。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节奏代谢的机制。 根据目前的模型，这个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/BMAL 1驱动“时钟”的转录， 蛋白质PERIOD（PER）1、PER 2、色氨酸（CRY）1和CRY 2，其与每个蛋白质相互作用 其他抑制CLOCK/BMAL 1的活性，从而抑制其自身的合成。同样的 转录机制也驱动许多所谓的“时钟控制基因”的节律性表达。 最终导致全身组织的许多输出节律。其中一个钟- 控制基因是夜曲蛋白，我实验室的一个重点。我们已经证明，在小鼠中这种基因的缺失 导致对饮食引起的肥胖的抵抗力，改变胆固醇和甘油三酯代谢的节律 并增加对炎症挑战如LPS的抵抗力。虽然Nocturnin蛋白是 作为RNA酶家族（CCR 4去腺苷酸酶）的一员，我们最近已经证明， Nocturnin的底物不是RNA，而是NADP（H）。Nocturnin是一种NADP（H）磷酸酶， 将NADP（H）转化为NAD（H）。在这项提案中，我们试图使用新的小鼠模型， 为了了解夜曲蛋白的组织特异性作用， 同种型（一种线粒体，另一种主要结合于细胞内膜）。我们还将携带 我们进行了代谢组学和代谢通量实验，以确定 NADP（H）和NAD（H）水平。此外，我们将验证小分子调节剂的夜曲蛋白 我们已经在高通量筛选中鉴定出了这些分子，并将使用这些分子来干扰Nocturnin 在细胞和小鼠中的活性。我们在核心时钟的中央机制方面的工作也将在年继续进行。 建议的融资期限。我们已经解决了CLOCK/BMAL 1的晶体结构， 这些数据允许鉴定进化上保守的 整个蛋白质的功能域，并揭示了更多的见解的机制， 这些蛋白质运作并设定昼夜节律周期。未来五年，我们将扩大 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.