Characterization of core clock complexes in time and space using quantitative proteomics

生物钟是一种细胞内源性、自我维持的振荡器，几乎存在于人体的每个细胞中，在细胞和组织生理学中发挥着重要作用。生物钟允许我们的器官从分子上预测和准备一天中与我们的自行车环境相关的经常性需求。驱动这些振荡的分子机制包括转录和翻译反馈环。在过去的几年里，基于MS的定量蛋白质组学在昼夜生物学中的应用拓宽了我们对蛋白质丰度和功能的动态如何有助于细胞和代谢过程的日常调控的理解。这项技术补充和扩展了以前建立的转录学研究，并为我们对分子和细胞时钟的知识提供了一个关键的新角度。除了表达蛋白质组学之外，基于MS的相互作用蛋白质组学与亲和纯化方法相结合，对鉴定在时钟分子机制中起关键作用的新的相互作用因子做出了巨大贡献。然而，这一机制仍有许多未知的方面，例如转录因子复合体BMAL1/CLOCK如何以时间和组织依赖的方式调节启动子的结合。我们建议将最先进的MS技术与定量蛋白质组学相结合，以最终了解BMAL1/CLOCK转录复合体如何在时间上但也在组织特定的背景下调节转录。具体地说，这项资助的目的是研究BMAL1/CLOCK复合体与染色质结合的时间和空间组成，以及细胞和小鼠组织的细胞质中的BMAL1/CLOCK复合体的时间和空间组成，以识别和功能表征新的辅助调节因子。许多影响昼夜节律的过程，如蛋白质的稳定性和蛋白质之间的相互作用，都受到翻译后修饰(PTM)的调节。因此，通过利用基于MS的定量蛋白质组学，这一强大的技术不仅可以识别PTM，还可以表征它们的时间动力学，这项资助还旨在识别从细胞和组织分离的时钟蛋白中新的振荡翻译后修饰，并表征它们的功能作用。总之，这项拨款的工作将阐明BMAL1/时钟复合体的时间和空间调制，这两个方面都是昼夜节律功能的关键方面。

Circadian clocks are cell endogenous self-sustainable oscillators found in virtually every cell in the body that play a fundamental role in cellular and tissue physiology. Circadian clocks allow our organs to molecularly anticipate and prepare for recurrent needs across the day associated to our cycling environment. The molecular mechanisms driving these oscillations comprise transcriptional and translational feedback loops. In the last years, the application of MS-based quantitative proteomics to circadian biology has broadened our understanding of how dynamics of protein abundance and function contribute to the daily regulation of cellular and metabolic processes. This technology complements and extends previously established transcriptomic studies and provides a critical novel angle in our knowledge of the molecular and cellular clocks. In addition to expression proteomics, MS-based interaction proteomics in combination with affinity purification methods has greatly contributed to the identification of novel interactors with key roles in the clock molecular mechanism. Nonetheless, there are still many unknown aspects of this mechanism, for example how the binding of the transcription factor complex BMAL1/CLOCK to promoters is regulated in a temporal and tissue dependent manner. We propose here to apply state of the art MS technologies in combination with quantitative proteomics to ultimately understand how the BMAL1/CLOCK transcriptional complex can modulate transcription in a temporal manner but also in a tissue specific context. Specifically, this grant aims to study the temporal and spatial composition of BMAL1/CLOCK complexes bound to chromatin but also in the cytoplasm of cells and mouse tissues to identify and functionally characterize novel co-regulators. Many processes contributing to circadian timing such as protein stability and protein-protein interaction are regulated by posttranslational modifications (PTMs). Thus, by taking advantage of MS-based quantitative proteomics, a powerful technique not only to identify PTMs but also to characterize their temporal dynamics, this grant also aims to identify novel oscillating post-translational modifications in clock proteins isolated from cells as well as tissues and characterize their functional role. Together, the work of this grant will shed light on temporal and spatial modulation of BMAL1/CLOCK complexes, both critical aspects for circadian function.