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.

大多数生物都表现出生理学，代谢和行为的每日周期，所谓的昼夜节律是由昼夜节律产生的。正常昼夜节律的破坏，例如由于突变，喷射滞后或转移工作严重损害了人类的生理，例如，由于班位工人中癌症和代谢综合征的增加而揭示了人类的生理。 〜24小时的周期是通过基因调节的负反馈回路产生的，该回路具有暂时性调节的合成，翻译后修饰，相互作用和时钟蛋白降解的相互作用。 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,共转移，细胞定位和稳定性，昼夜节律）。我们的研究将为MBMAL/MCLOCK复合物的MCRY依赖性转录抑制作用提供重要的机械见解，并将MPER2蛋白稳定性和细胞定位的调节。这些分子过程对于维持昼夜节律的24小时和时钟受调节的人类行为和生理学至关重要。此外，我们的结构将推进哭泣的化学探针和新型治疗代谢调节剂的设计。