Structure-function analyses of mCRY-mPER2 and mCRY-mBMAL1 complexes

mCRY-mPER2 和 mCRY-mBMAL1 复合物的结构功能分析

• 批准号: 246768937
• 负责人: Professorin Dr. Eva Wolf
• 金额: --
• 依托单位: Arbeitsgruppe Strukturelle Chronobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/246768937?language=en
• 关键词: Structure; function; analyses; mCRY; mPER2

Most organisms exhibit daily cycles of physiology, metabolism and behaviour, so called circadian rhythms, which are generated by circadian clocks. Disruption of the normal circadian (~ 24 h) cycle e.g. due to mutations, jet-lag or shift-work severely compromises human physiology, as revealed, for example, by increased incidences of cancer and metabolic syndrome in shift workers. The ~24 h period is generated by gene-regulatory negative feedback loops with a spatio-temporally regulated interplay of synthesis, posttranslational modifications, interactions and degradation of clock proteins. Here we propose to study two essential protein-protein complexes of the mammalian circadian clock, the Cryptochrome1/2-mPERIOD2 (mCRY-mPER2)- and the Cryptochrome1/2-mBMAL1 (mCRY-mBMAL1) complex, at multiple levels ranging from high-resolution structural insights to circadian phenotypes in vivo. Methods will include X-ray crystallography, protein interaction studies (e.g. analytical gel filtration, isothermal titration calorimetry, fluorescence polarization, pull-down, native polyacrylamide gels) as well as functional and phenotypic analyses in live cells (e.g. luciferase complementation, fluorescence-two-hybrid, co-immunoprecipitation, FRET, FRAP, cotransactivation, cellular localization and stability, circadian oscillations). Our studies will provide important mechanistic insights into the regulation of mCRY and mPER2 protein stability and cellular localization and into the mCRY-dependent transcriptional repression of the mBMAL/mCLOCK complex. These molecular processes are essential for maintaining the 24 h period of the circadian clock and of clock regulated human behaviour and physiology. Furthermore, our structures will advance the design of CRY chemical probes and of novel therapeutic metabolic regulators.

大多数生物体表现出生理、新陈代谢和行为的日常循环，即所谓的昼夜节律，这是由生物钟产生的。例如，由于突变、时差或倒班工作而扰乱了正常的昼夜节律(~24小时)周期，严重损害了人类的生理，例如，倒班工人的癌症和代谢综合征的发病率增加。~24小时周期是由基因调控的负反馈环与时钟蛋白的合成、翻译后修饰、相互作用和降解等时空调控的相互作用产生的。在这里，我们建议从高分辨率的结构洞察到体内的昼夜节律表型，在多个水平上研究哺乳动物生物钟的两个基本蛋白质-蛋白质复合体，即隐色1/2-mPERIOD2(mCRY-mPER2)-和隐色1/2-mBMAL1(mCRY-mBMAL1)复合体。方法将包括X射线结晶学、蛋白质相互作用研究(例如分析凝胶过滤、等温滴定量热法、荧光偏振、下拉、天然聚丙烯酰胺凝胶)以及活细胞的功能和表型分析(例如荧光素酶互补、荧光双杂交、免疫共沉淀、FRET、FRAP、共激活、细胞定位和稳定性、昼夜振荡)。我们的研究将为调节mCRY和mPER2蛋白的稳定性和细胞定位以及mCRY依赖的mBMAL/mCLOCK复合体的转录抑制提供重要的机制见解。这些分子过程对于维持生物钟的24小时周期以及由生物钟调节的人类行为和生理是必不可少的。此外，我们的结构将推动CREY化学探针和新型治疗性代谢调节剂的设计。