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，由生物钟产生。扰乱正常昼夜节律（约 24 小时）周期，例如由于突变，时差或轮班工作严重损害了人类的生理机能，例如，轮班工人癌症和代谢综合征发病率的增加就表明了这一点。大约 24 小时的周期是由基因调节负反馈环路产生的，其中时钟蛋白的合成、翻译后修饰、相互作用和降解的时空调节相互作用。在这里，我们建议研究哺乳动物生物钟的两种必需蛋白质-蛋白质复合物，Cryptochrome1/2-mPERIOD2 (mCRY-mPER2)-和Cryptochrome1/2-mBMAL1 (mCRY-mBMAL1)复合物，在从高-解析体内昼夜节律表型的结构见解。方法包括 X 射线晶体学、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）以及活细胞中的功能和表型分析（例如荧光素酶互补、荧光二-混合、免疫共沉淀、FRET、FRAP、共反式激活、细胞定位和稳定性、昼夜节律振荡）。我们的研究将为 mCRY 和 mPER2 蛋白稳定性和细胞定位的调节以及 mBMAL/mCLOCK 复合物的 mCRY 依赖性转录抑制提供重要的机制见解。这些分子过程对于维持 24 小时生物钟以及时钟调节的人类行为和生理至关重要。此外，我们的结构将推进 CRY 化学探针和新型治疗性代谢调节剂的设计。