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

大多数生物体表现出生理、代谢和行为的每日周期，即所谓的昼夜节律，其由生物钟产生。正常昼夜（~24小时）周期的破坏，例如由于突变，时差或轮班工作，严重损害人体生理学，如轮班工人中癌症和代谢综合征发病率增加所揭示的。~24 h周期是由基因调控负反馈回路产生的，具有时空调控的时钟蛋白的合成、翻译后修饰、相互作用和降解的相互作用。在这里，我们建议研究哺乳动物昼夜节律钟的两个基本蛋白质-蛋白质复合物，隐色素1/2-mPERIOD2（mPER-mPER2）-和隐色素1/2-mBMAL1（mPER-mBMAL1）复合物，在多个层次上，从高分辨率的结构见解，在体内的昼夜节律表型。方法将包括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、荧光双杂交、免疫共沉淀、FRET、FRAP、共转录、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生物钟的24小时周期和生物钟调节的人类行为和生理是必不可少的。此外，我们的结构将推进CRY化学探针和新型治疗代谢调节剂的设计。

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.