Functional & biochemical characterisation of circadian timekeeping mechanisms in mammalian cells

功能性

• 批准号: 1857414
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1857414
• 关键词: Functional; biochemical; characterisation; circadian; timekeeping

Daily rhythms underlie the biochemistry, physiology and behavior of terrestrial life. In humans, the 24-hour cycle is inextricably linked to patterns of sleep and wake, blood cortisol levels, cognitive function and the expression of around 20% of the genome in each cell. Dysregulation of these rhythms is associated with metabolic diseases and increased cancer susceptibility. In addition, the emerging field of chronopharmacology aims to take advantage of the daily changes of physiology and metabolism in order to maximize the therapeutic effect of drugs.Circadian rhythms at the cellular level are regulated by networks of transcriptional, translational and post-translational factors. It is widely accepted that the cellular clockwork is driven by a core transcriptional feedback mechanism; the main elements being activating transcription factors CLOCK and BMAL1, and their repressors: Period (PER) and Cryptochrome (CRY). CRY is regarded as the main repressor, although PER is required for its full function, and so CRY mutants have been used as arrhythmic animal and cellular models. However, the O'Neill lab have observed persistent, albeit less robust, circadian rhythms in cells from mice that are homozygous null for CRY1 and CRY2 - reported by PER2::LUC bioluminescence. We find that these CRY-independent PER2::LUC rhythms are regulated by post-translational mechanisms, such as phosphorylation by casein kinase , that also contribute to timekeeping in wild type cells. This project will build upon these prior findings by identifying the key molecular components that sustain timekeeping in both wild type and CRY-deficient cells, though pharmacological manipulation and tandem mass spectrometry. Furthermore it will aim to characterize the function of these pathways using cutting edge tools for genetic manipulation such as inducible expression and CRISPR-Cas9 genome editing, in order to determine the minimal set of clock components that are necessary and sufficient to sustain cellular circadian rhythms. This knowledge will reveal novel therapeutic targets for metabolic diseases and cancers, as well as opening new avenues for chronopharmacology.

日常节律是陆地生命的生物化学、生理学和行为的基础。在人类中，24小时周期与睡眠和觉醒模式、血液皮质醇水平、认知功能以及每个细胞中约20%基因组的表达有着千丝万缕的联系。这些节律的失调与代谢疾病和癌症易感性增加有关。此外，时间药理学的新兴领域旨在利用生理和代谢的日常变化，以最大限度地提高药物的治疗效果。细胞水平的昼夜节律受转录，翻译和翻译后因子网络的调节。人们普遍认为，细胞时钟是由核心转录反馈机制驱动的;主要元件是激活转录因子CLOCK和BMAL 1，以及它们的阻遏物：Period（PER）和Cryptochrome（CRY）。CRY被认为是主要的阻遏物，尽管PER是其全部功能所必需的，因此CRY突变体已被用作哺乳动物和细胞模型。然而，奥尼尔实验室已经观察到持续的，虽然不太稳健，昼夜节律的细胞从小鼠是纯合子无效的E11和E12-报告的PER 2：：LUC生物发光。我们发现，这些非依赖性PER 2：：LUC节律是由翻译后机制调节的，例如酪蛋白激酶的磷酸化，这也有助于野生型细胞的计时。该项目将建立在这些先前的研究结果，通过药理学操作和串联质谱法，确定在野生型和β-缺陷细胞中维持时间保持的关键分子组分。此外，它的目标是使用遗传操作的尖端工具来表征这些途径的功能，例如诱导型表达和CRISPR-Cas9基因组编辑，以确定维持细胞昼夜节律所必需和足够的最小时钟组件集。这些知识将揭示代谢性疾病和癌症的新治疗靶点，并为时间药理学开辟新的途径。

项目成果

CRYPTOCHROMES promote daily protein homeostasis.

• DOI: 10.15252/embj.2021108883
• 发表时间: 2022-01-04
• 期刊: The EMBO journal
• 作者: Wong DCS;Seinkmane E;Zeng A;Stangherlin A;Rzechorzek NM;Beale AD;Day J;Reed M;Peak-Chew SY;Styles CT;Edgar RS;Putker M;O'Neill JS
• 通讯作者: O'Neill JS

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology.

• DOI: 10.1038/s41467-021-25942-4
• 发表时间: 2021-10-15
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Stangherlin A;Watson JL;Wong DCS;Barbiero S;Zeng A;Seinkmane E;Chew SP;Beale AD;Hayter EA;Guna A;Inglis AJ;Putker M;Bartolami E;Matile S;Lequeux N;Pons T;Day J;van Ooijen G;Voorhees RM;Bechtold DA;Derivery E;Edgar RS;Newham P;O'Neill JS
• 通讯作者: O'Neill JS

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

补偿性离子转运缓冲每日蛋白质节律，以调节渗透平衡和细胞生理学

• DOI: 10.1101/2020.05.28.118398
• 发表时间: 2020
• 作者: Stangherlin A

CRYPTOCHROME suppresses the circadian proteome and promotes protein homeostasis

• DOI: 10.1101/2020.05.16.099556
• 发表时间: 2020-05
• 期刊: bioRxiv
• 作者: David C. S. Wong;Estere Seinkmane;Alessandra Stangherlin;Aiwei Zeng;N. Rzechorzek;Andrew D. Beale

CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

• DOI: 10.15252/embj.2020106745
• 发表时间: 2021-04-01
• 期刊: The EMBO journal
• 作者: Putker M;Wong DCS;Seinkmane E;Rzechorzek NM;Zeng A;Hoyle NP;Chesham JE;Edwards MD;Feeney KA;Fischer R;Peschel N;Chen KF;Vanden Oever M;Edgar RS;Selby CP;Sancar A;O'Neill JS
• 通讯作者: O'Neill JS