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

使用定量蛋白质组学表征核心时钟复合体的时间和空间特征

• 批准号: 428041612
• 负责人: Professorin Dr. Maria S. Robles Martinez, Ph.D.
• 金额: --
• 依托单位: Institut für Medizinische Psychologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/428041612?language=en
• 关键词: Characterization; core; clock; complexes; time

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.

昼夜节律钟是细胞内源性自我维持振荡器，几乎存在于体内的每个细胞中，在细胞和组织生理学中发挥着重要作用。昼夜节律时钟使我们的器官能够从分子角度预测并为一天中与骑行环境相关的经常性需求做好准备。驱动这些振荡的分子机制包括转录和翻译反馈回路。在过去的几年中，基于质谱的定量蛋白质组学在昼夜节律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促进细胞和代谢过程的日常调节的理解。该技术补充和扩展了先前建立的转录组学研究，并为我们对分子和细胞时钟的了解提供了一个重要的新颖角度。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，极大地促进了在时钟分子机制中起关键作用的新型相互作用物的鉴定。尽管如此，该机制仍有许多未知的方面，例如转录因子复合物 BMAL1/CLOCK 与启动子的结合如何以时间和组织依赖性方式受到调节。我们在此建议将最先进的 MS 技术与定量蛋白质组学相结合，以最终了解 BMAL1/CLOCK 转录复合物如何以时间方式以及组织特异性背景下调节转录。具体来说，这项资助的目的是研究与染色质以及细胞和小鼠组织的细胞质中结合的 BMAL1/CLOCK 复合物的时间和空间组成，以识别和功能表征新型协同调节因子。许多影响昼夜节律的过程，例如蛋白质稳定性和蛋白质-蛋白质相互作用，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（一种强大的技术，不仅可以识别 PTM，还可以表征其时间动态），这项资助还旨在识别从细胞和组织中分离的时钟蛋白中的新型振荡翻译后修饰，并表征其功能作用。总之，这项资助的工作将揭示 BMAL1/CLOCK 复合体的时间和空间调制，这都是昼夜节律功能的关键方面。