Proteomic Identification Of Circadian Clock Core Protein BMAL1-Interacting Ubiquitin Ligases And Their Regulation On BMAL1 Metabolism

Background and aims: Physiological and behavioral activities in almost all organisms display cyclical rhythms in sync with the environment, known as biological rhythms. Circadian clocks and human health are closely related. Biological clock disorders seriously affect the physiological and behavioral rhythms of organisms, resulting in endocrine disorders, disrupted homeostasis, and other symptoms. Seven clock-related genes, such as Per1, Per2, Per3, Cry1, Cry2, Clock and Bmal1, have been discovered and cloned in mammals. These genes and their protein products constitute the independent regulation of transcription and translation feedback loop, which is the molecular mechanism of the biological clock. The further study of the regulatory mechanism of the biological clock proteins by post translational modification is of importance in revealing the nature of the life rhythm activity. Therefore, in this thesis, on the one hand, we discovered an E3 ligase interacting with the core protein, BMAL1, of the biological clock through the protein interaction network analysis, and explored its effect on the biological rhythm and the underlying mechanism. On the other hand we used a spectral counting-based label-free quantitative proteomic approach to identify BMAL1-interacting proteins and to study their influence on BMAL1 metabolism, and to further explore their regulation of biological rhythms.Methods: Through protein interaction network analysis, we discovered a potential BMAL1-interacting E3 ligase, TRAF2. We verified the interaction between TRAF2 and BMAL1 and explored the mechanism of its regulation on BMAL1 through affinity purification and Western blotting analysis. We further used qPCR to measure the rhythm changes in N2 a cells after TRAF2 knockdown. We expressed FLAG-BMAL1 in HEK293 T cells, isolated BMAL1-interacting proteins by immunoprecipitation, digested them with trypsin to obtain peptides for mass spectrometry identification of BMAL1-interacting proteins followed by immunoblotting validation. We further discovered the regulatory mechanism of BMAL1 through a series of biochemical experiments.Results: The E3 ligase TRAF2 screened from the protein interaction network platform has a role in the regulation of biological rhythms. TRAF2 could reduce BMAL1 stability through the enhancement of BMAL1 ubiquitination. Knockdown of TRAF2 could alter the biological rhythm in cells. We also identified 216 BMAL1-interacting proteins through a spectral counting-based label-free quantitative proteomic approach. Through the bioinformatic analysis, we found that BMAL1-interacting proteins have important biological functions in the ubiquitin dependent proteasome process, nuclear translocation, protein localization, etc. E3 ligases UBR5 and STUB1/CHIP could reduce BMAL1 stability through promoting BMAL1 ubiquitination. Knockdown of a deubiquitinating enzyme USP9 X could decrease BMAL1 stability.Conclusion: The E3 ligase TRAF2 regulates the biological rhythm through regulating BMAL1 ubiquitination and stability. E3 ligases UBR5 and STUB1 downregulate BMAL1 through the ubiquitin-proteasome pathway. Their functions on the regulation of the biological rhythm need to be further explored and validated. These findings provide a basis for further functional studies. The further study of the regulatory mechanism of the biological clock proteins by the ubiquitin-proteasome pathway is important in revealing the nature of the rhythm activity.